MMNO．H2O在花粉和孢子外壁產生的效應

The Effect of 4-Methylmorpholine N-oxide Monohydrate (MMNO · H2O) on Pollen and Spore Exines

將垂枝樺、玻璃苣、加蘆那杜鵑、歐洲水青岡、麝香百合和歐洲赤松的成熟花粉粒及石松的孢子暴露在80°C的MMNO·H2O下，處理時間由30分鐘至310分鐘。30分鐘後小通道的直徑大約從25nm擴張為50nm，一般通道的大小在發育期間大概是50nm。除加蘆那杜鵑外，觀察其他種類花粉在處理近五個小時後的變化。玻璃苣和歐洲赤松在花粉外壁形態上所產生的變化，與我們先前研究中所指出在外壁發育晚期才添加的孢粉質有關。花粉外壁發育的早期，花粉質堆積在部份細胞膜表面的覆蓋物上（即網叢，外壁單元結構），也就是孢粉質接受粒上。在外壁發育的晚期，次級孢粉質堆積於網叢之間或是它們的表面。這個階段中，孢粉質接受粒會交叉連結遍及整個外壁上。我們的推測是MMNO·H2O對於多醣類就像是一種有效的溶劑，可經由網叢內的小通道和網叢交叉連結之間滲入外壁中。一般的穿透式電子顯微鏡樣品製作過程中，由於花粉暴露在水和溶劑中使多醣類被移除，而引起外壁組成產生裂縫。垂枝樺和歐洲水青岡的花粉在MMNO·H2O中所產生的外壁變化，同樣被推測可能因為溶劑的侵蝕是平行於網叢核心和它的次級結構單位。麝香百合花粉外壁上大多數較細長的柱狀體明顯是由一個網叢所形成，被侵蝕後露出外層的纏繞物。石松孢子經處理後所顯露的是較緊密的間隔，大約是10nm，周圍則呈薄層狀排列，且聚集為線狀結構。這些線狀結構在橫切面中觀察為環狀，直徑變化較大（約100-200nm）。使用原子力和隧道式掃描電子顯微鏡進行觀察後，推測大部分長的次級單位呈螺旋狀。同樣地，可以觀察到在結構間是放射狀交叉連結的型式。相較下，花粉外壁沒有產生變化的種類，可能必須近一步觀察這些花粉的外壁內層。我們的推測是：花粉外壁內層因為網叢填塞緊密，也沒有一些孢粉素的次級堆積，因而較外壁外層具有較大的抗性。

Mature pollen grains of Betula pendula, Borago officinalis, Galluna vulgaris, Fagus sylvatica, Lilium longiflorum, Pinus sylvestris and spores of Lycopodium clavatum were exposed to 4-Methlymorpholine N-oxide monohydrate (MMNO·H2O) at 80°C for times varying from 30 minutes to 310 minutes. After 30 minutes microchannnels had expanded from approximately 25 nm to 50 nm in diameter, the channel size during development is approximately 50 nm. After about 5 hours in MMNO·H2O pronounced changes in exine morphology were observed except in Calluna. Changes in exine morphology of Borago and Pinus involved sporopollenin that our previous studies indicated had been added late in development. Early in development sporopollenin accumulates on Sporopollenin Acceptor Particles (SAPs) that are part of the plasma membrane surface coating (tufts, the unit-structures of exines). Subsequently, late in development, secondary sporopollenin is added between tufts or on their surfaces. At this stage SAP cross linkages permeate the exine. Our interpretation is that MMNO·H2O, as a potent solvent for polysaccharide, penetrates the exine through the core zone (microchannels) of tufts and tuft cross linkages. When polysaccharide is removed exposure to water and solvents normally used for TEM preparation cause fracture of exine components. Exine changes during MMNO·H2O in Betula and Fagus may be similarly interpreted since erosion parallels the orientation of the tuft core and its subunits. In Lilium the most slender columellae consisting apparently of one tuft were eroded enough to expose coils of outer (binder) zone. In Lycopodium the MMNO·H2O resulted in exposure of closely spaced (approximately 10nm) circumferentially oriented lamellae that are grouped into linear structures. These linear structures are circular in cross section and greatly variable in size (most are 100-200 nm in diameter). Scans using atomic force- and scanning tunnelling- microscopy suggest that the mainly longitudinal subunits appear to be helical, and radial cross linkages between the structures can also be discerned. No change, other than enlargement of microchannels, could be seen in the endexines of the species studied. Our interpretation is that the greater resistance of the endexine than the ectexine is due to close packing of tufts without any secondary deposition of sporopollenin.