蝴蝶蘭於氮、磷、鉀養分逆境下之生長反應與基因功能分析

Abstract

蝴蝶蘭 (Phalaenopsis spp.) 原生於熱帶雨林，具有特化的氣生根可附著於樹幹表面。蝴蝶蘭的根部的外皮層具根被包覆，內皮層則有特化的通過細胞協助水分及養分的吸收，但其表面並無根毛。因蝴蝶蘭特殊的著生習性及根部構造，本研究之目的在探討養分受限環境下蝴蝶蘭對營養元素的吸收及分配，以瞭解蝴蝶蘭在低養分環境的生存機制。 本研究於氮、磷及鉀養分逆境下瞭解蝴蝶蘭之生長反應並進行基因功能分析，試驗以Phalaenopsis Sogo Yukidian ‘V3’為材料，施用含不同濃度氮 (0、0.71、7.14 mM)、磷 (0、0.16、1.61 mM) 及鉀 (0、0.38、3.84 mM)之液態肥料，栽植於30/25 oC人工氣候室進行觀察。施肥處理八週後，缺氮、缺磷及缺鉀處理對植株營養生長之影響不大，僅地上部鮮重隨磷肥施用濃度的下降而降低，但觀察成熟葉及根部之氮、磷及鉀的濃度已有顯著降低，顯示蝴蝶蘭對養分的反應緩慢。施肥處理八週後，將植株由30/25 oC移至25/20 oC之自然光照室進行低溫催花。其中以低氮(0.71 mM) 處理組最早抽梗、植株達到50% 抽梗率所需天數最少 (47天) ，全株葉色轉為淺綠， SPAD讀值下降；缺磷處理抽梗延遲，於低溫處理80天後，其抽梗率僅達33.3%，葉尖呈紫紅色；而缺鉀處理組達50% 抽梗率所需天數 (74天) 較對照組 (56天) 長，於花朵開放後其上位葉出現黃色斑塊及壞疽，病徵由葉尖往基部擴散。自花梗發育後，缺氮及缺磷處理之新葉生長速率明顯下降，地上部生長受抑制；但新葉生長速率於缺鉀處理與正常施肥組無異。施肥處理32週後，植體中氮、磷及鉀大量被運移至花梗及花朵，由於花梗發育耗用大量養分，致使營養缺乏之影響較易於生殖生長期中顯現。 短期養分缺乏對蝴蝶蘭的外觀及生長並無明顯影響。藉由生物晶片檢測施肥處理八週後蝴蝶蘭的成熟葉部及根部之基因表現，發現缺氮、缺磷、缺鉀的在基因層次上具有相同調節的基因，在成熟葉中，缺氮、缺磷及缺鉀會共同誘導794個上調及129個下調基因；而在根部則有101個上調及89個下調基因。而在缺乏氮、磷及鉀的處理下，葉片中DNA、RNA調控、蛋白質合成及降解及受體激酶之基因表現皆受缺肥誘導而增加，顯示短期養分缺乏時，蝴蝶蘭已有相應調控，協助植體內的磷之分配與利用。對應阿拉伯芥缺磷之研究顯示蝴蝶蘭的葉及根部之磷轉運蛋白 (phosphate transporter)、磷酸水解酶 (phosphatase) 及甘油類化合物sulfoquinovosyldiacylglycerol 2 (SQD2)等基因上調，且影響糖基甘油酯 (glycosylglycerides) 之代謝。顯示在短期養分缺乏下，蝴蝶蘭雖於外觀無明顯之缺肥徵狀顯現，但於基因調控層次上已有反應，其中轉錄因子PATC128122於蝴蝶蘭之葉片及根部皆受缺磷誘導而表現量大量增加，此基因未來可作為蝴蝶蘭缺磷訊號標誌基因。

Phalaenopsis is an epiphyte native to tropical broadleaf forest in Taiwan. Epiphytic roots attach to the surface of tree trunks instead of growing in soil. Unique structures and morphology of Phalaenopsis roots include velamen outside epidermis, absence of root hairs, lack or absence of branch roots and passage cells in endodermis. Understanding the mechanism of nutrients uptake and metabolism in Phalaenopsis is important to determine how efficient nutrition usage is achieved under intermittent supply. In this study, the growth response and gene expression of Phalaenopsis under N, P, or K deficiency stress were investigated. Phalaenopsis Sogo Yukidian ‘V3’ was grown in a 30/25 oC phytotron and fertilized with solutions containing different concentrations of N (0, 0.71, or 7.14 mM), P (0, 0.16, or 1.6 mM), and K (0, 0.38, or 3.84 mM). Except for lower fresh weight observed in P deficiency treatments, N, P, or K deficiency had little effect on vegetative growth after 8 weeks of treatment, despite lower concentrations of N, P, and K in mature leaves and roots. This shows that Phalaenopsis responds slowly to fertilization. After 8 weeks of treatment in 30/25 °C, plants were transferred to 25/20 °C phytotron to induce spikes. Plants given low N concentration (0.71 mM) had earliest spiking, and required least number of days to 50% spiking (47 days). The SPAD value decreased and the color of the entire foliage of these N-deficient plants turned light green. Phosphorus deficiency delayed spiking where only 33% spiking rate was reached after 80 days of low temperature induction. Purple color was evident on the leaf tips of P-deficient plants. Plants deficient in K required longer to reach 50% spiking (74 days) compared with control (56 days). Yellow spots and necrosis occurred on the upper leaves of these plants after flower opening, and the symptom developed from the tip to the base of the leaves. New leaf growth decreased significantly after spike development in N- and P-deficient plants, but not in K-deficient plants, which had similar new leaf growth rate to plants given full N, P, and K fertilization. After 32 weeks of starvation treatment, significant amounts of N, P, and K were transported to spike and flowers. Because a large amount of nutrients was required for the inflorescence development, the symptoms of nutrition deficiency became evident during the reproductive stage. Microarray analysis revealed differential gene expression in mature leaf and roots during 8 weeks of starvation. There were 794 up-regulated and 129 down-regulated genes commonly appearing in shoots and 101 up- and 89 down-regulated genes in roots under N, P, or K deficiency stress. The transcript levels of DNA and RNA regulation, protein degradation and synthesis, and signaling of receptor kinases were increased in mature leaves. Comparison of the expression of P starvation in Arabidopsis and Phalaenopsis, the phosphate transporter, phosphatase, SQD2 were up-regulated in leaves and roots, and effect on the glycosylglyceride biosynthesis in leaves and roots. Under short-term P starvation, there were no significant symptom in phenotype but the gene regulation already changed. During short-term nutrient starvation, there was regulation of genes for survival in nutrition stress. The PATC128122 was highly up-regulated both in leaf and roots; hence this gene may be used as a marker gene under P starvation.