臭氧對植物花粉萌發、花粉管生長、蛋白質及微細構造之影響

Abstract

本研究主要在探討，臭氧對靜止中葵百合花粉萌發率和其蛋白質中胺基酸組成的影響，以及臭氧對活化中葵百合花粉萌發率、花粉管長度、花粉管外部形態及內部微細構造，和蛋白質中胺基酸組成的影響。 在靜止和活化中的花粉，經臭氧處理後，其花粉萌發率皆隨著臭氧濃度的升高，而有明顯的降低。與未經臭氧處理之花粉萌發率比較：靜止中的花粉在0.04ppm低濃度臭氧下處理後，花粉萌發率為46.5％，降低了22.7％；1.21ppm高濃度臭氧處理後，花粉萌發率只有14.3％，降低了54.8％。活化中的花粉，在0.04ppm低濃度臭氧處理後，花粉萌發率為49.5％，降低了23.6％；在1.21ppm高濃度臭氧處理後，花粉萌發率只有20.0％，降低了53.2％，其花粉管長度亦減少了50％。此表示，靜止中的花粉和活化中的花粉一樣，對臭氧具有相似的敏感度，臭氧對花粉活力，萌發能力及花粉管發育有明顯抑制作用。 以LM、SEM和TEM顯微觀察，臭氧處理後之花粉管外部形態及內部微細構造。花粉管前端透明區受到流動細胞質嚴重幹擾，或完全由流動細胞質所取代，而產生膨大或破裂之現象。管內液胞分佈遍及花粉管中段，甚至在花粉管前端亦可觀察到液胞。在0.04ppm低臭氧濃度下，即可發現，花粉管壁外圍呈非平滑圓柱狀；在1.21ppm高臭氧濃度下，嚴重受損之花粉管細胞質呈非連續狀，管壁直徑粗細不均。在SEM下發現，管壁有穿孔產生，紋路變異成鱗片狀或網狀紋。在TEM下發現：經臭氧處理的花粉管壁橫切面呈不規則狀，在1.21ppm高臭氧濃度下，花粉管前端外壁異常加厚，中後段內外管壁變薄，基部液胞區管壁嚴重變形。管壁有質離之現象，在花粉管的細胞膜上，出現許多在電子顯微鏡下，染色較深的點狀物質。花粉管細胞質內的澱粉體和粒線體，皆隨臭氧濃度提高，其瓦解面積和膜系變異增加。 分析臭氧處理下，靜止中花粉蛋白質之胺基酸組成，發現十七種胺基酸中，有十一種胺基酸比值下降。而活化花粉其花粉管內十三種胺基酸比值下降。此外，天冬胺酸（aspartate）和麩胺酸（glutamate）兩種胺基酸，無論在靜止或活化之花粉內，其比值反而上升。 在臭氧馴化濃度下，花粉管蛋白質中除甘胺酸（glycine）和脯胺酸（proline）比值下降外，其他有十四種胺基酸的比值上升，其中以丙胺酸（alanine）、精胺酸（arginine）和纈胺酸（valine）的上升輻度最大。有九種蛋白質含量提高，其中包含三種分子量為67KD者，以及兩種45KD者、三種43KD者和一種14KD者；四種蛋白質相對合成速率降低，包含一種分子量為65KD者、兩種64KD者和一種50KD者。此與1.21ppm臭氧濃度下，胺基酸比值下降，合成受到抑制之現象並不相同。 本試驗之另一材料「水稻」，在1ppm臭氧濃度下，以不同時間處理水稻各栽培種發現：其六種栽培種中，以梗稻對臭氧最敏感，秈稻次之，糯稻最輕微。 本研究可供「花粉在臭氧環境下之生理機制」研究之基本資料。並且依此尋找對臭氧具敏感性的花粉種類，做為監測臭氧的生物指標。

Both rest and activated pollen of lily Star Gazer were exposed to various concentration of ozone. The stress effect of ozone was evaluated through viability testing germination rate, protein changes and ultrastructure disorganization. Additionally six different cultivars of rice pollen are exposed to ozone during rest. This study claims that lily Star Gazer can be used as a bioindicator for ozone contamination and provides primary information that can be utilized in the study of physical mechanisms of pollen in an ozone environment. Star Gazer pollen has similar levels of sensitivity during both rest and germination. When Star Gazer pollen is exposed to ozone during rest, as the ozone concentration increases, there is an evident decrease in the rate of pollen germination. Exposed to 0.04ppm ozone during rest, the germination rate was 46.5%, which was 32.8% lower than the control. Give 1.21ppm ozone, the germination rate was decreased to 14.3%, which was 80% lower than the germination rate of the control group. During germination, the germination rate of stargazer pollen exposed to 0.04ppm of ozone was 49.5%, which was 32.3.% lower than the control group's germination rate. The germination rate of stargazer pollen exposed to 1.21ppm of ozone during germination was just 20.0%, 72.0% lower than the germination rate of the control group. According to observations made with light microscope, the clear zone located at the tip of the pollen tube is severely obstructed by streaming cytoplasm, of the tip of the pollen tube is totally replaced by the streaming cytoplasm and swells or bursts as a result. The distribution of the tip of the vacuole can be found in the central section of the pollen tube and even in the tip of the pollen tube. The pollen tubes of the stargazer pollen group exposed to 1.21ppm of ozone were not cylindrical and were of unequal thicknesses. The cytoplasm if the pollen tubes were also discontinuous. With the scanning electron microscope, perforations in the tube wall were discovered. Scale and web-like lines were also founds on the surface of the pollen tube walls with the scanning electron microscope. By analyzing cross-sections with the trans-electron microscope, it was discovered that the outer wall of the pollen tube had a non-circular shape. The wall at the tip of the pollen tube in the stargazer pollen exposed to 1.21ppm of ozone was abnormally thick, while the wall of the central section was abnormally thin. The shape of the outer wall of the vacuolate zone at the base of the pollen tube was extremely changed in comparison with the control group. A high density of electrons also appeared on the plasma membrane of the pollen tube. Autolysis of the surface area of the amyloplasts and abnormal changes in the membrane cristae of the mitochondria increased as the concentration of ozone increased. There were seventeen different kinds of amino acids analyzed in the stargazer pollen during the dormant stage in this study. Out of these seventeen amino acids, the rate of amino acid content in fourteen of these amino acids decreased. In the germination stage, the rate of content of the amino acid structure in the cytoplasm also decreased. In addition, the rate of content of glutamic acid and aspartic acid increased during both the dormant stage and the germination stage. It is highly possible that the composition of amino acids was inhibited by the presence of ozone. As a higher percentage of ozone was gradually added to the stargazer pollen grain during germination, the rate of amino acid content increased for fifteen of the seventeen amino acids with alanine, arginine, and valine showing the largest increases; glycine and proline showed decreases in their rate of amino acid content. The protein content of nine kinds of protein increased. There were three kinds of protein with a molecular weight of 67KD, two kinds with a molecular weight of 45KD, three kinds with a molecular weight of 43KD, and one kind with a molecular weight of 14D. The relative rate in the protein content of four of the proteins decreased with one of the proteins having a molecular weight of 65KD, two kinds with a molecular weight of 64KD, and the last with a molecular weight of 50KD. When the results from stargazer pollen exposed to 1.21ppm of ozone during germination are compared with above results, the rate of amino acid concentration fell and the composition of protein were uninhibited. In a related experiment, six different pollens of cultivar from Oryza sativa sativa were exposed to 1.00ppm of ozone. Among the six cultivars, japonica was the most sensitive to ozone, while glutinous was the most tolerant.