過去在營養缺乏所誘導的植物細胞自噬研究中，以探討缺乏碳和氮源為主，但缺磷誘導自噬的研究甚少。本研究將探討阿拉伯芥自噬相關基因與植物磷酸恆定之關聯性。先前文獻已指出大量表現轉錄因子 PHOSPHATE STARVATION RESPONSE 1 (AtPHR1) 或液泡磷酸膜轉運蛋白 PHOSPHATE TRANSPORTER 5;1 (AtPHT5;1) 能活化植物缺磷反應基因。因此，我們利用菸草短暫表達系統大量表現 AtPHR1 和 AtPHT5;1 產生缺磷反應來觀察自噬相關基因 AtATG8f 在菸草表皮細胞中的表現與分佈型態。實驗結果顯示，在大量表現 AtPHR1 情況下，GFP-AtATG8f 呈現均勻表現在細胞質的形態。而大量表現 AtPHT5;1 情況下，細胞質中的 GFP-AtATG8f 受到缺磷反應影響，由均勻分布轉為零星的點狀訊號，可能是自噬作用活性提升使 GFP-AtATG8f 呈現自噬體的形態。我們推測大量表現 AtPHT5;1 造成細胞內產生缺磷反應對活化細胞自噬的影響較為直接。另一方面，我們觀察到阿拉伯芥轉殖株35S:GFP-AtATG8f 在缺磷和缺氮環境下，自噬體也能大量累積在成熟區、延長區和細胞分裂區三個根部發育區域。由於 GFP-AtATG8f 蛋白在根毛區域大量聚集，我們推測自噬體參與根毛生長發育或是吸收、回收營養的過程。我們將目前已知 AtATG8f 上的結合位點：LC3 docking site (LDS) 和 UIM docking site (UDS)，同時進行點突變(Y50,A,L51A,I77A,F78A,I79A)，並利用三分子綠螢光技術來探討阿拉伯芥AtATG8f 與泛素結合酶 PHOSPHATE 2 (AtPHO2) 和磷酸轉運蛋白 PHOSPHATE 1 (AtPHO1) 之間的交互作用。結果顯示 AtPHO1 和 AtATG8f 之間沒有交互作用，而 AtPHO2 與正常或具突變位 AtATG8f 皆有交互作用，表示 AtPHO2 可能參與細胞自噬，但其扮演的角色還需更深入探討。

Most studies of plant nutrient deficiency-induced autophagy focused on carbon (C) and nitrogen (N) source, but little is known about phosphate (Pi) limitation-induced autophagy. In this study, we aimed to explore the involvement of the Arabidopsis autophagy-related genes (AtATGs) in plant Pi homeostasis. We overexpressed two Pi homeostasis-related genes, the Arabidopsis transcription factor PHOSPHATE STARVATION RESPONSE 1 (AtPHR1) and the vacuolar PHOSPHATE TRANSPORTER 5;1 (AtPHT5;1) in tobacco leaves by agroinfiltration. When AtPHR1 was overexpressed, GFP-AtATG8f localized in the cytosol; when AtPHT5;1 was overexpressed, GFP-AtATG8f showed a punctate pattern, which might result from increased formation of autophagosomes. AtPHT5;1 overexpression likely decreased the cytosolic phosphate level and thus induce autophagy. In the GFP-AtATG8f transgenic plants, a large number of autophagosomes were observed in the differentiation and elongation zones, and the apical meristem of roots under Pi and N deficiency, particularly in root hairs. We surmise that autophagosomes may participate in the growth and development of root hairs or their role in nutrient absorption and recycling. In addition, we generated a AtATG8f mutant variant with defective LC3 docking site (LDS) and UIM docking site (UDS) to investigate its interaction with the other two Pi homeostasis-related proteins, PHOSPHATE 1 (AtPHO1) and PHOSPHATE 2 (AtPHO2). The tripartite split-GFP association analysis showed that both the wild-type AtATG8f and mutated AtATG8f can interact with AtPHO2 but not AtPHO1, indicating that AtPHO2 may be involved in the regulation of autophagy.

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