印度梨形孢菌共生小白菜之代謝體學研究及文心蘭高溫誘導油菜醇影響開花機制之探討

Abstract

內生真菌印度梨形孢真箘（Piriformospora indica or P. indica）的共生效益已經在各種宿主植物中被證明與促進生長發育及逆境之耐受性有關。然而，在代謝體學研究中是罕見的。透過使用高通量氣相質譜儀，在共生與非共生的小白菜根中鑑定出1126種代謝化合物，當中的549種代謝物已被鑑定。分析結果顯示，當宿主植物與P. indica共生後，其多數代謝物和代謝途徑將被 reprogram。當中γ-氨基丁酸（GABA），oxylipin-family compounds,，poly-saturated fatty acids, auxin 及其中間物在共生根中被高度誘導產生及de novo synthesized。相反地，nicotinic acid和dimethylallylpyrophosphate 其合成則大量減少。在外施化合物的 In vivo 試驗中證實 ，GABA 能夠引發植物對病原體的免疫力，並增強宿主植物對於高鹽以及高溫之耐受性。此外，由 nicotinic acid 刺激所產生的活性氧/氮物的產生會被Piriformospora indica 抑制，以增加共生的成功率。代謝體學研究以及與共生相關之特異性代謝物的鑑定有助於了解P. indica是如何賦予宿主植物共生效益。 南西文心蘭具有獨特的開花過程，其中涉及了持續的熱誘導氧化壓力和內源性氧化還原的改變。然而，相關信號的傳導參與，如促進此熱誘導的氧化壓力開花之植物激素與生理機制仍尚未釐清。在本研究中，我們調查了在低溫（20 ºC）及高溫（30 ºC）環境下生長的文心蘭轉錄組，發現除了氧化壓力和熱反應的上調基因外，油菜素類固醇（BR）相關基因亦參與其中。透過qRT-PCR分析，發現OgPIF4和OgPIL6以及六個油菜素類固醇相關基因（OgBZR1，OgBRH1，OgDWF1，OgBR6Ox，OgBRI1 和 OgBAK1） 的表現量也被上調。施加 ep-brassinolide （ep-BL）於文心蘭上，證實BR信號傳導可促進蘭花提早開花和花序的快速發育。此外，無論在低溫或高溫條件下施加 ep-BL 後，文心蘭開花基因如 OgFT、OgLFY 和 OgAP1 之表現量均有提高的現象。藉由在阿拉伯芥野生型和 pifq 突變株中大量表現 OgBZR1 基因，證明OgDWF1藉由OgBZR1和熱誘導的 PIF4 調節開花和 BR 的生合成。綜合以上實驗結果，說明了BR信號與PIF4的結合可以促進 BR 之生合成，並與 ASA-GSH 氧化還原協同作用，促使文心蘭提早開花。

Root colonization by endophytic fungus Piriformospora indica facilitating growth/development and stress tolerance has been demonstrated in various host plants. However, global metabolomic studies are rare. By using high-throughput gas- chromatography-based mass spectrometry, 549 metabolites of 1,126 total compounds observed were identified in colonized and uncolonized Chinese cabbage roots, and hyphae of P. indica. The analyses demonstrate that the host metabolomic compounds and metabolite pathways are globally reprogrammed after symbiosis with P. indica. Especially, γ-amino butyrate (GABA), oxylipin-family compounds, poly-saturated fatty acids, and auxin and its intermediates were highly induced and de novo synthesized in colonized roots. Conversely, nicotinic acid (niacin) and dimethylallylpyrophosphate were strongly decreased. In vivo assays with exogenously applied compounds confirmed that GABA primes plant immunity toward pathogen attack and enhances high salinity and temperature tolerance. Moreover, generation of reactive oxygen/nitrogen species stimulated by nicotinic acid is repressed by P. indica, and causes the feasibility of symbiotic interaction. This global metabolomic analysis and the identification of symbiosis-specific metabolites may help to understand how P. indica confers benefits to the host plant. Oncidium Gower Ramsey hybrid orchid possesses an exceptional flowering process that involves persistent thermal–induced oxidative stress and endogenous redox alteration. Despite this, the participation of other signaling, such as phytohormone aiding this persistent thermal–induced oxidative stress flowering, is less understood. In this work, we survey two solexa transcriptomic libraries of genes transcribed respectively under low ambient (20 oC) and high ambient temperature (30 oC) incubation in Oncidium orchid. In addition to up-regulated genes encoding for oxidative-stress and thermal responses, brassinosteroid (BR) –responsive genes are prominent as well. Through qRT- PCR, thermal responsive genes such as OgPIF4 and OgPIL6 along with six BR- responsive genes namely OgBZR1, OgBRH1, OgDWF1, OgBR6Ox, OgBRI1 and OgBAK1 are up-regulated as well. Exogenous application of ep-brassinolide (ep-BL) confirms the function of BR-signaling by promoting Oncidium early flowering and rapid inflorescence development. Moreover, three Oncidium flowering integrator genes, such as OgFT, OgLFY, and OgAP1 are elevated after such exogenous treatment under both low and high –ambient temperatures. Functional analyses by over-expressing OgBZR1 in Arabipdopsis wild-type and pifq mutant lines, demonstrate the regulation of OgDWF1 expression through OgBZR1 and thermal-induced PIF4 to mediate flowering and de novo BR-biosynthesis. Altogether, our results fairly exemplify BR-signaling coupled with PIF4 can promote de novo BR-biosynthesis, and synergistic to Oncidium’s ASA-GSH redox hub, induces early flowering.