細胞分裂素生合成基因對子葉不等大堇蘭分生組織異位的維持角色

Abstract

苦苣苔科的堇蘭(Streptocarpus rexii)不具有頂端分生組織，其細胞分裂活力位於葉子上，使葉子取代頂端分生組織的功能而具有生長新器官與維持植株發育的能力。過去研究顯示堇蘭這種異位的分生組織會受到外加細胞分裂素(cytokinin)的促進，而這和模式植物中細胞分裂素負責分生組織活力的維持，具有相似的性質。故我們嘗試解開細胞分裂素是如何參與堇蘭異位分生組織活力的調控。本研究自堇蘭分離出細胞分裂素生合成異戊烯基轉移酶基因(ISOPENTENYLTRANSFERASE, IPT) ，並檢測其在堇蘭幼苗的分生組織中的表現模式及是否受到外加植物荷爾蒙的調控。我們自堇蘭分離出五個IPT同源基因(SrIPT1, SrIPT2, SrIPT3, SrIPT5, SrIPT9)，其中SrIPT1與SrIPT3集中表現在花或根部，SrIPT5和SrIPT9則在各個器官都有表現，而SrIPT2具有提前終止密碼子，可能是一個假基因(pseudogene)。我們更進一步發現SrIPT1, SrIPT5與SrIPT9均大量表現在堇蘭的異位分生組織內(groove meristem和basal meristem)，而SrIPT5在分生組織處(葉子基部)的表現量比不具有分生組織活力分佈的葉尖端的表現量還高。這些結果支持異位分生組織的活力維持需要高濃度細胞分裂素的假設。最後，我們發現外加的荷爾蒙如生長素與吉貝素會抑制SrIPT3, SrIPT5, SrIPT9表現，而細胞分裂素會促進SrIPT1, SrIPT3與SrIPT9表現，暗示植物荷爾蒙調節SrIPT的表現量可能與堇蘭異位分生組織的維持有關。我們的研究證實IPT基因在堇蘭的分生組織內的表現，顯示其可能參與維持分生組織的活力。這也進一步暗示細胞分裂素生合成可能和堇蘭的分生組織自莖頂異位至葉子後的活力維持有關。

Streptocarpus rexii (Gesneriaceae) lacks the conventional shoot apical meristem (SAM). On the other hand, unorthodox meristems form on the leaves of S. rexii, and are capable of producing lateral organs and maintaining plant’s growth. Previous researches indicated that the exogenous cytokinin (CK) treatment affected the activities of the unorthodox meristems of S. rexii. We therefore aimed to understand how CK is involved in maintaining the unorthodox meristem activity of S. rexii. In this study, I isolated the CK biosynthesis IPT gene (ISOPENTENYLTRANSFERASE) and examined their expression patterns in relation to meristem activity in the seedling of S. rexii. I also tested whether the expression levels of SrIPT genes can be altered by exogenous hormone treatments in order to understand whether SrIPTs can be regulated by major phytohormones. Five IPT homologous genes were isolated from S. rexii (SrIPTs): SrIPT1, SrIPT2, SrIPT3, SrIPT5, and SrIPT9, while SrIPT2 is perhaps a pseudogene because of the formation of a premature stop codon. Real-time PCR of the other four SrIPTs revealed that SrIPT1 mainly expressed in floral organs and SrIPT3 mainly in roots, and SrIPT5 and SrIPT9 expressed in all organs examined. Furthermore, I found the strong expression signals of SrIPT1, SrIPT5 and SrIPT9 within the unorthodox meristems (basal and groove meristems) via RNA in situ hybridization. I also discovered that SrIPT5 strongly expressed in meristem-located (proximal end) rather than no-meristem-located (distal end) lamina tissue of older leaves. These results supported the hypothesis that meristem-localized CK level is required for the maintenance of these unorthodox meristems. Exogenous hormone treatments such as auxin and gibberellin can inhibit certain SrIPTs expression but CK increase their expression instead, implying that hormone regulated SrIPTs expression may be important for meristem maintenance. Overall, our results indicated that certain CK biosynthesis IPT genes expressed in unorthodox meristems, suggesting they have a role on meristem maintenance.