Loss of Anisocotyly in European Gesneriaceae and Its Evolutionary Consequence

子葉不等大是苦苣苔科植物 (Gesneriaceae)特有的現象，在種子發芽後，其中一片子葉會藉由分生組織持續生長而成為大子葉，另外一片子葉則無法持續性的生長而形成小子葉。這樣子葉不等大的特性可能可以幫助幼苗適應陰暗的環境。其中，舊世界苦苣苔科類群發展出極為特殊的子葉不等大現象，而新世界苦苣苔科類群則保持子葉等大的特性。特別的是舊世界苦苣苔科中歐洲苦苣苔成員 (Ramonda myconi, Haberlea rhodopensis)喪失子葉不等大的現象而成為子葉等大的幼苗。這些喪失子葉不等大的種，對比於仍具備子葉不等大的近緣種，提供了我們得以比較研究苦苣苔科子葉不等大發育機制的轉化及其演化上的影響。我們藉由比較歐洲苦苣苔成員及仍具備子葉不等大發育的模式物種菫蘭 (Streptocarpus rexii)間的發育及形態差異，經由子葉面積大小的測量，歐洲苦苣苔成員兩片子葉呈現相似的生長速率，因此維持兩片子葉等大的特徵，然而菫蘭的大小子葉的確具有不同的生長速率，使得菫蘭兩片子葉異速生長並造成菫蘭子葉不等大的特性。進一步檢測子葉的細胞大小、毛狀物的分佈、及分生組織活性檢測均顯示歐洲苦苣苔科成員的兩片子葉在發育早期已停止生長，兩片子葉間也無形態上明顯的差異，同時也無法觀察到持續的分生組織活性刺激子葉持續生長。前人研究結果顯示與分生組織活性有關的基因如Class I KNOX (STM, BP)可能參與在舊世界苦苣苔子葉不等大的形成，我們也取得舊世界苦苣苔物種及其他具代表性的苦苣苔科成員的KNOX基因並進行分析，冀望未來能進一步檢測特定的KNOX基因序列是否與子葉等大與子葉不等大轉換有關，然而結果並未顯示任何關聯性，可能是因為取樣尚不足。未來針對上述基因進行表現及功能性研究相信能更加了解KNOX基因在子葉不等大現象扮演的角色。最後，本篇研究依據細胞分裂、細胞擴增、毛狀物分佈及側脈形成與否，指出歐洲苦苣苔成員為子葉等大類群Anisocotyly is common in Gesneriaceae, which refers to that both cotyledon are unequally developed. The macrocotyledon can continuously grow while microcotyledon ceases to grow after cotyledon expansion. Anisocotyly might be able to help seedlings to adapt/survive in dense shade environments. Unlike other dicot plants, Old World species evolve anisocotylous trait during germination while New World species remain isocotyly instead. Within Old World European Gesneriaceae species (Ramonda myconi and Haberlea rhodopensis), there appears to be a secondary loss of anisocotylous trait back to isocotyly. These isocotylous species thus provide a great opportunity to investigate how anisocotyly develop, and loses during Gesneriaceae evolution. In this study, I compared cotyledon development in isocotylous European Gesneriaceae species to Streptocarpus rexii, their sister clade species with true anisocotyly. The results of cotyledon size measurement showed that European Gesneriaceae species exhibit nearly equal cell growth rate and thus maintain both cotyledons in the same size. But in Streptocarpus rexii, growth rate between macrocotyledon and microcotyledon was different thus altered cotyledon allometry growth. Further examinations of cell size, trichome distribution and meristematic activity failed to find any differences between cotyledons of European species. All observations showed that both cotyledons of European Gesneriaceae species ceased to grow in early stages and thus no prolonged meristematic activity could be observed. Previous studies indicated that meristematic activity gene such as Class 1 KNOX (STM, BP) may play a role in the formation of anisocotyly. I therefore isolated KNOX gene homologues from several Gesneriaceae species in the hope to find the correlation of transition of isocotyly and anisocotyly to any corresponding KNOX homologues evolution. The results so far did not indicate any correlation perhaps due to the lack of detailed samplings. Further expression and functional studies on these genes will be helpful to understand their roles on anisocotyly. Nonethesless, this study indicated that European Gesneriaceae species is isocotylous because of no differential cell division, cell expansion, trichome distribution and lateral vein formation between both cotyledons. The species phylogeny indicates European isocotylous species are perhaps a loss of anisocotyly.致謝................................................................................................................................I文摘要.......................................................................................................................IIbstract.......................................................................................................................IVontent........................................................................................................................VI. Introduction..........................................................................................................1.1 Anisocotyly in Gesneriaceae..................................................................................1.2 Isocotyly in European Gesneriaceae......................................................................4.3 Meristematic activity in cotyledon expansion........................................................6.4 KNOX members as candidate genes......................................................................8.5 Anisocoyly in Old World Gesneriaceae..................................................................10.5 Aims of this study..................................................................................................11. Materials and Methods.........................................................................................13.1 Plant materials.......................................................................................................13.2 Germination observation.......................................................................................13.3 Fixation..................................................................................................................14.4 Tissue clearing.......................................................................................................14.5 Scanning electron microscopy (SEM)...................................................................16.6 Cell division observation.......................................................................................16.7 RNA extraction......................................................................................................17.8 First strand cDNA synthesis..................................................................................18.9 Polymerase Chain Reaction (PCR)........................................................................19.10 TA Cloning...........................................................................................................21.11 Transformation.....................................................................................................21.12 Phylogenetic analysis...........................................................................................23. Results.....................................................................................................................25.1 Germination process..............................................................................................25.2 Morphological observation of cotyledons.............................................................26.3 Distribution of cell divisions on cotyledons..........................................................28.4 Phylogeneitc analysis of KNOX in Gesneriaceae..................................................29. Discussion................................................................................................................32.1 Morphological difference between isocotylous and anisocotylous species...........32.2 Roles of cell division in cotyledon expansion........................................................34.3 Mechanism and explanation for anisocotyly loss...................................................36.4 Candidate genes for transition of anisocotyly/isocotyly........................................40. Future Aspects........................................................................................................43. References...............................................................................................................44. Figures and Tables.................................................................................................49ig 1. Germination process of S. rexii..........................................................................49ig 2. Germination process of R. myconi.....................................................................50ig 3. Germination process of H. rhodopensis............................................................50ig 4. Area ratios between macrocotyledons and microcotyledons n different stage of seedlings............................................................................52ig 5. The cotyledon area data of three species in different stage of seedlings...........53ig 6. SEM of R. myconi seedlings..............................................................................54ig 7. SEM of H. rhodopensis seedlings......................................................................55ig 8. Three types trichomes........................................................................................56ig 9. Cell division signals from Aniline Blue Staining..............................................57ig 10. Reconstruction of STM using Neighbor-Joining criteria.................................59ig 11. Reconstruction of STM using Neighbor-Joining criteria.................................60ig 12. Diagram of cotyledon expansion.....................................................................61ig 13. Simplified Gesneriaceae phylogeny................................................................62ab 1. Species list for Class 1 KNOX phylogenetic analysis.......................................63ab 2. Nested design ANOVA (analysis of variance) of epidermal ell size (μm2) on cotyledons among each species...........................................64ab 3. Cell plate density on cotyledons among species...............................................66. Appendices..............................................................................................................67.1 Cell plate density between proximal region and distal region...............................67.2 Different trichome distribution on cotyledons of R. myconi..................................68.3 Different trichome distribution on cotyledons of H.rhodopensis...........................69.4 Different trichome distribution on cotyledons of S. rexii.......................................70.5 Average epidermal cell size of each species..........................................................71.6 Alignment of STM..................................................................................................72.7 Alignment of BP.....................................................................................................73.8 Clearing of first true leaves and cotyledons...........................................................74.9 SEM of seedlings with previous studies................................................................75.10 Aniline Blue Staining photo of R. myconi............................................................76.11 Aniline Blue Staining photo of H. rhodopensis....................................................77.12 Aniline Blue Staining photo of S. rexii.................................................................78.13 Genera with anisocotyly feature...........................................................................7