以高效質譜分析平台研究植物免疫調控胜肽並探討相對應之訊息傳遞機制

Abstract

訊息胜肽已知在多細胞生物體內扮演一重要調節者，其可透過細胞間溝通去調控生物體內之多樣化生理反應。然而植物目前被發現之訊息胜肽數量相當有限，主要原因為植物內生性訊息胜肽通常在植物體內含量很低並且需要經過特定蛋白酶酵素的活化才能從前驅蛋白上被切出來，尤其是防禦功能相關之胜肽。所以，找尋此類防禦性胜肽需使用大量的植物組織且胜肽的偵測被侷限在特定生物活性試驗之選擇。為了克服偵測訊息胜肽的種種困難，我們在此研究中建立了一個高通量定量胜肽體學方法來找尋更多新的植物防禦性訊息胜肽。此方法結合了我們自製序列資料庫來進行內生性胜肽序列鑑定並且利用質譜定量方法來搜尋在植物受傷後可能被誘導的防禦性胜肽。在此研究中除了已知的受傷訊息胜肽systemin之外，同時也在番茄葉中找到數個新發現的受傷誘導胜肽。其中一個被鑑定出的新胜肽為植物當中免疫指標蛋白PR-1所衍生出之胜肽片段，PR-1又屬於CAP蛋白家族一員，所以我們稱此胜肽為CAP-derived peptide 1 (CAPE1)。此胜肽被進一步確認為僅在受傷後的番茄葉中大量表現並且可調控番茄的免疫反應產生抗病及抗蟲之效果。此研究提出一個有效率的方法找尋植物內生性防禦胜肽，並且首次證明未知功能蛋白PR-1在免疫訊息傳遞上所扮演的角色。除此之外，PR-1在其他生物也具有高度保留性，我們測試在阿拉伯芥中預測之PR-1衍生胜肽也同樣具有生物活性，此結果顯示此胜肽將可以廣泛應用於增強不同植物抗性以對抗各種外來環境壓力。

Signaling peptide is known to be an important mediator for cell-cell communications to regulate the diverse physiological responses in multicellular organisms. However, in plants, only a limited number of signaling peptides have been identified. This may due to the plant bioactive peptides are low abundant and dynamically derived from precursor by activation of protease, especially for defense functions. Thus, the most plant defense signaling peptides were identified by using a large quantity of plant tissues for peptide discovery and the peptide detection was limited by the selection of bioassay. In an attempt to overcome the challenges in identifying plant signaling peptides, we developed a high-throughput quantitative peptidomics approach for the discovery of novel defense signaling peptide. This approach integrated the hypothetical peptide database and a quantitative MS approach to profile and discover wound-induced defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel wound-induced peptides were confidently identified and quantified in Solanum lycopersicum (tomato). One of novel wound-induced peptides was derived from the pathogenesis-related 1 protein (PR-1) of CAP superfamily, termed as CAP-derived peptide 1 (CAPE1). This peptide was further confirmed to be significantly induced by wounding and found to trigger immune response for both antipathogen and antiherbivore activities in tomato. This study is not only proposed an efficient way to detect defense signaling peptide in plants but also highlights a new role for PR-1 in immune signaling. As PR-1 is highly conserved across many organisms and the putative peptide from AtPR1 was also found to be bioactive in Arabidopsis, our results suggest that the homologues peptides of CAPE1 may be useful for enhancing resistance to various stresses in other plant species.