大白花蝴蝶蘭‘V3’於養分逆境下的生理反應及缺磷下的基因表現

Abstract

蝴蝶蘭(Phalaenopsis spp.)為附生蘭，對營養逆境有極高的耐受度，缺肥徵狀不易顯現。為了瞭解蝴蝶蘭在養分缺乏下的生存機制，本論文分為三大部分，分別從生理、解剖與基因表現層面，探討必要大量元素氮、磷或鉀缺乏逆境對蝴蝶蘭的影響。 本研究給予大白花蝴蝶蘭Phal. Sogo Yukidian ‘V3’中苗不同濃度的氮(0、0.71、7.14 mM)、磷(0、0.16、6.14 mM)、鉀(0、0.38、3.84 mM)肥料，調查蝴蝶蘭於缺乏養分逆境下的病徵與生長反應。蝴蝶蘭栽種於30/25 oC的人工氣候室，缺肥4週、8週或12週的植株於外觀及生長量無差異。而植株栽種於高溫環境下維持營養生長且缺肥處理32週後，缺氮植株鮮重降低，葉面積及葉數減少；缺磷植株有落葉現象；缺鉀則沒有任何徵狀。移至25/20 oC的環境進行低溫催花的植株，缺肥處理32週後，缺氮植株有乾重降低、葉片變黃、抽梗提早現象，但花朵數減少、花徑變小且花梗縮短；缺磷植株抽梗率降低、落葉明顯且葉片呈紫色；缺鉀未影響植株外觀及生長量，僅造成抽梗延後。另外，於營養生長下缺氮12週即造成蝴蝶蘭光合作用效率下降，然而，即使缺鉀32週後，植株的光合作用能力仍不受影響；缺磷32週處理下雖不影響葉綠素螢光參數Fv/Fm，但最大二氧化碳交換速率下降。 蝴蝶蘭Phal. Sogo Yukidian ‘V3’的根部外層具有2層特化的根被組織，內皮層和外皮層則分別具有細胞壁呈現O型及U型加厚的長細胞，以及通過細胞座落其中，內皮層中的通過細胞位於木質部的相對處，這些構造可能有利於水分及養分的吸收及保留。磷為植物生長與繁衍所必須之元素，缺磷不會改變蝴蝶蘭根部與葉片的細胞形態，但缺磷造成蝴蝶蘭根部中柱變小、維管束數量減少。 自微矩陣分析中挑選37個於缺磷下明顯表現改變的基因，利用反轉錄聚合酶連鎖反應加以驗證，大部分葉片中的上調基因可以被驗證，但下調基因無法被確認。其中，PATC128122蛋白質上具RING功能區塊，對於缺磷具專一性反應，並顯著於缺磷12週後在蝴蝶蘭的葉片和根部內提高表現，原位雜交結果顯示其表現位置為根部的皮層細胞、葉片主脈的韌皮部。利用含兩倍花椰菜嵌紋病毒35S啟動子的載體大量表現PATC128122於阿拉伯芥內，發現35S::PATC128122轉殖株栽種於不同磷濃度(250、50、10 mM Pi)的條件下，其轉殖株的鮮重、無機磷濃度與外觀皆與野生型植株沒有差異。 綜合上述結果顯示，蝴蝶蘭的缺肥徵狀於長期缺肥32週下顯現，又以開花植株最為嚴重。但於相對短期的缺磷逆境下，縱使植株外觀未見明顯變化，基因表現及維管束形態已產生差異，顯示蝴蝶蘭植株內部已啟動因應機制，未來將可再進一步探討蝴蝶蘭於缺肥下表現基因的功能。

Phalaenopsis spp., an epiphytic orchid, is tolerant to nutrient stresses. The nutrient deficient symptoms usually develop very slowly. To understand how Phalaenopsis survives under nutrient starvation, we examined the effect of nitrogen (N), phosphorus (P) or potassium (K) deficiency on Phalaenopsis from the aspects of physiology, anatomy, and gene expression, respectively. To investigate the symptoms and growth responses of Phalaenopsis under nutrient deficiency, Phal. Sogo Yukidian ‘V3’ plants were supplied with fertilizer containing different concentrations of N (0, 0.71, 7.14 mM), P (0, 0.16, 6.14 mM), or K (0, 0.64, 6.4mM). Plants were grown in a 30/25 oC phytotron, and no significant differences in plant appearance and growth were observed during 4, 8, or 12 weeks of starvation. When plants were kept at vegetative growth and straved for 32 weeks, N deficiency resulted in reduced whole-plant fresh weight, total leaf area, and leaf number and P deficiency aggravated leaf falling. However, no effects were observed under K deficiency. The 8-week nutrient-straved plants were further subjected to low temperature (25/20 oC) to provoke flowering. At 32 weeks of nutrient deficiency, N deficient plants showed early spiking, reduced dry weight, and appearance of yellow leaves; P deficiency caused decreased spiking rate along with the increase of falling leaves and the development of purple pigment. However, the growth were not affected by K deficiency except for delayed spiking. Besides, photosynthesis efficiency decreased significantly after 12 weeks of N deficiency but was not affected by K deficiency even after 32 weeks of deficiency. While low-P treatment reduced the net carbon dioxide exchange rate, it did not affect chlorophyll fluorescence (Fv/Fm), suggesting photosystem II was not impaired. There are two cell layers of velamen at the outermost of Phal. Sogo Yukidian ‘V3’ roots. There are long cells with O-shaped walls and U-shaped walls in endodermis and exodermis, repectively. Passage cells were among endodermis and exodermis and opposite to the xylem in endodermis. These structures may be beneficial for water and nutrient uptake and retention. Anatomical analyses revealed that P deficiency did not signigicantly affect cell morphology of roots and leaves but decreased the size of stele and the number of vascular bundle in roots. Furthermore, several P starvation-responsive genes identified from a previous microarray analysis were selected for validation by reverse transcription polymerase chain reaction analysis. I was able to validate the up-regulated expression for most of genes in leaf but unable to confirm the down-regulated expression of several genes. Among them, the expression of PATC128122, encoding a RING domain-containing protein, was highly up-regulated in the leaves and roots subjected to 12-week P deficient treatment but didn’t respond to N or K deficient treatment. Results of in situ RNA hybridization indicated its expression in the root cortex and leaf phloem. Transgenic Arabidopsis thaliana overexpressing PTAC128122 driven by 35S promoter of Cauliflower mosaic virus displayed no differences in fresh weight, inorganic phosphate concentration, and visible appearance in comparison with wild-type plants grown under various phosphate supplies, 250, 50, and 10 μM phosphate. In summary, in spite of no noticeable changes during vegetative growth, the expression of a subset of genes and vascular pattern of Phalaenopsis are altered, likely as adaptative responses to cope with limited P. Of note, the long term nutrient starvation during the vegetative growth severly affects the flowering of Phalaenopsis at the later stage.