Characterization and functional identification of rice pollen-specific OsCPK and its potential downstream substrate

Abstract

水稻中有二十九個 CDPKs (calcium dependent protein kinase) 基因成員，其中七個專一表現於花粉。先前實驗室發現將 OsCPK26 大量表現於水稻花粉，會產生雄不稔性狀，另外，若大量表現於百合花花粉，則會強烈抑制花粉萌發，萌發率僅 11.5%，但 OsCPK26(G2A) 卻均無上述現象。為了找出 OsCPK26 抑制花粉萌發的關鍵性胺基酸或蛋白質結構，利用暫時性大量表現 OsCPK26-ECFP 融合蛋白於百合花花粉，發現附膜是 OsCPK26 抑制花粉萌發的必要非充分條件。於 domain swapping 和 C-terminal serial-deletion 實驗中，發現OsCPK26 僅需蛋白 N 端的 7 個胺基酸便可附膜，且至少需其 1-59 a.a.片段，才具有抑制能力，最後透過 point mutation 和 gain-of-function 策略，證實 46-58 a.a.間的 α-helix 為抑制花粉萌發的重要結構。 儘管鈣離子訊號在花粉萌發及延長上扮演重要的角色，但目前只找到少數花粉專一的 CDPKs 之下游受質蛋白。為了尋找下游受質蛋白，本實驗利用 pull-down親和力純化受質蛋白，並以 LC-MS/MS 鑑定其身分，獲得 OsCPK29 之候選受質蛋白，於眾多可能的蛋白中，選擇粒線體的 ATP synthase β-subunit 做進一步研究，將之命名為 ATPSB1。雖然 ATPSB1 普遍存在於水稻各組織中，但 ATPSB1 的相對轉錄量與蛋白表現量，皆於花粉中最高。本實驗發現 ATPSB1 基因靜默水稻會產 生花粉發育不全和花藥無法開裂之性狀，最後導致稔實極低 (約 1%)，另外，暫時性表現 ATPSB1-EYFP 融合蛋白於百合花花粉，可知其分布於粒線體中，令人驚訝的是，會附膜的 OsCPK29 和 ATPSB1 共表現時，出現 ATPSB1 從細胞質轉移到細胞膜的現象，暗示這兩個蛋白質間可能有交互作用。進一步將 ATPSB1 與 α-subunit(ATPSA1) 共表現，則發現 ATPSA1 與 ATPSB1 行為相似，均與 OsCPK29 結合於細胞膜，但其他同樣會附膜的 OsCPK21 與 OsCPK26 則均無上述現象。這些結果 說明 ATPSB1 可能於花粉發育或萌發上扮演重要角色，且可能OsCPK29 之下游受質蛋白，未來可更進一步調查 ATPSB1 於細胞膜上之生化特性與生理角色。

In rice, CDPK (calcium dependent protein kinase) genes are constituted by twenty nine members with seven of them expressed predominantly in pollen. Previous studies revealed that overexpression of the pollen-predominant OsCPK26, but not its non-membrane bound G2A form, caused male-sterility in rice and strongly inhibited the germination rate of lily pollen to be less than 11.5%. We characterized the protein motifs and amino acid residues in OsCPK26 critical for the inhibition effect. By tracing the transiently overexpressed OsCPK26-ECFP fusion protein in lily pollen, we found that membrane-binding is a prerequisite but is not sufficient for the inhibition effect. The domain-swapping and C-terminal serial deletion constructs revealed that even the N-terminal 7-a.a. fragment is enough for the membrane-binding of OsCPK26. However,the minimal length for the inhibition effect of OsCPK26 require at least its 1-59 a.a.fragment. Through point mutation and gain-of function strategies, an α-helix reside between 46-58 a.a. region was demonstrated to be critical. Although calcium signaling is known to play critical roles in pollen germination and tip growth, only a few proteins had been identified as substrates downstream to pollen-predominant CDPKs so far. Using affinity pull-down, candidate substrate proteins bound by OsCPK29 were identified by LC-MS/MS. A mitochondrial ATP synthase β-subunit, named as ATPSB1, was chosen for further study. Although ATPSB1 is expressed ubiquitously, relatively high transcript and protein level were detected in the mature pollen grains than all other tissues. Silence expression of ATPSB1 disrupted pollen development and anther dehiscence, finally resulted in an extremely low fertility rate (~1%). Transient expression of EYFP-fused ATPSB1 in lily pollen revealed its location within mitochondria. Surprisingly, co-expression of the membrane-bound OsCPK29 recruited ATPSB1 onto the plasma membrane, suggest a strong interaction between these two proteins. Moreover, the α-subunit of ATP synthase (ATPSA1) was also co-distributed with the beta subunit and OsCPK29. None of the other membrane-bound OsCPKs, including OsCPK21 and OsCPK26, possess the affinity with ATP synthase. These results suggest that the ATPSB1 play important roles in pollen development/germination and ATPSB1 may be a native downstream substrate for OsCPK29 in pollen. The functional relevance of ATP synthase on the plasma membrane require further investigations.