以蛋白質體學研究授粉與2,4-D處理之玉米穎果早期發育之蛋白質表現

Abstract

生長素（auxin）在穎果發育中是重要的調控物質，參與調控細胞分化與細胞擴大的功能。本研究利用蛋白質體分析技術，比較處理生長素類調節劑2,4-D與正常授粉的玉米穎果之蛋白質體表現差異，探討生長素在玉米穎果發育中的分子作用機制。 本試驗以玉米品種台南22號為試驗材料，栽培於田間或溫室中，在玉米雌穗花絲尚未吐出前予以套袋，再分別進行授粉或2,4-D處理，另外以未授粉也未處理2,4-D的穎果做為對照組。在處理或授粉之後1天、3天、5天、9天、12天取穎果，測定鮮、乾重、水分含量、葡萄糖與蔗糖含量、醇溶性蛋白質含量與SDS-PAGE分析，並進行蛋白質雙向電泳分析。從建立不同發育時期之玉米穎果的總蛋白質體圖譜，與經由2,4-D處理、授粉和對照組的相互比較，找出差異表現的蛋白質，並探討這些蛋白質參與生長素作用於穎果發育之生理功能。 試驗的結果顯示，2,4-D處理可誘導玉米穎果發生類似單性結果（parthenocarpic-like）的生長現象。處理2,4-D的玉米穎果在早期（1到5天）的鮮乾重、水分含量以及糖類（葡萄糖和蔗糖）的增加較正常授粉的穎果更為快速，但在處理5天之後，處理2,4-D之玉米穎果的各項生理現象漸漸停止。由形態的觀察顯示，auxin雖可促進果皮和珠心組織的生長，但沒有胚及胚乳組織發生的現象；處理2,4-D的玉米穎果中也無法偵測到胚乳細胞分化的指標，亦即澱粉和儲藏性蛋白質zein的累積。這暗示著2,4-D可能促進玉米穎果的早期生長，使穎果成為一個明顯的積儲（sink），快速累積光合作用產物；但單只有生長素的作用尚無法使玉米穎果發育成一個正常且成熟的種子，也顯示有其他的授粉訊息在調控穎果的發育。 以蛋白質雙向電泳技術分析2,4-D處理和授粉的玉米穎果蛋白質體表現，發現在第3天之後，開始有germin-like protein 1的表現，且此蛋白質在正常授粉和2,4-D處理的穎果中皆有表現，對照未授粉的穎果，推論此蛋白質應是受到auxin所誘導，參與在穎果細胞擴大的過程中；在5天之後，有三個蛋白質在膠片上被觀察到，分別是calmodulin、glycine-rich RNA binding protein 1和profilin。此三個蛋白質，如同germin-like protein1的表現，都隨正常授粉和2,4-D處理的穎果生長而表現，故此三者應該也是和auxin的作用相關。其中，calmodulin可能與auxin的訊息傳導有關，profilin則可能是因細胞內的鈣離子濃度變化，參與細胞支架的重組，至於glycine-rich RNA binding protein 1在本試驗中，只能推測其可能受到auxin所誘導，其實際作用與功能仍未知。此外，正常授粉的穎果隨著時間上的發育，開始有澱粉和儲藏性蛋白質的累積，而處理的穎果則沒有，可見上述4個蛋白質並非穎果胚乳發育成熟的關鍵蛋白質，應該還有其他的授粉訊息非經由auxin的途徑，使穎果得以生長分化，此部分還需進一步的試驗證明。另外，在授粉12天之穎果蛋白質雙向電泳中，糖解相關酵素表現量大幅增加，推測是為提供旺盛細胞活動所需之能量。在未授粉也未處理2,4-D之對照組穎果，在發育時間第5天時，有chitinase A和22K antifungal protein大量表現，可能參與在未授粉穎果之衰化過程中，保護其不受病害感染有關。 本試驗中，初步建立的早期玉米穎果發育蛋白質雙向電泳圖譜，已有40個蛋白質被確定身份，其中33個為授粉12天之穎果表現的蛋白質，其他7個為對照組第5天所特定表現的蛋白質。以授粉12天之穎果來說，佔大多數的是代謝類的蛋白質，其次是和訊息傳遞相關的蛋白質以及抗氧化酵素，這些都是未來探討玉米穎果發育之分子機轉的有用的資訊。

Auxin is one of key regulators for physiological processes during maize caryopsis growth and development, including cell division, cell enlargement, and dry matter accumulation. The main objective of this study was to study the molecular basis of auxin’s function on maize caryopsis development, using proteome analysis technique. A maize cultivar (Tainan 22, TN22) was grown in the field or greenhouse. At silking, maize ears were either treated with a synthetic auxin 2, 4-D or hand pollinated. And kernels which were not treated with 2, 4-D and handed - pollination were used as the controls for comparison. Kernels were sampled at 1, 3, 5, 9, and 12 days after treatment or pollination, and than stored at -80℃ for analysis. Changes of fresh weight, dry weight, water content, sugar content, alcohol soluble protein, and starch staining pattern were measured. To determine the expression patterns of maize kernel proteins, a high resolution two - dimensional gel electrophoresis was used. Differential expressed proteins were further sampled and identified by ESI-Q-TOF MS. The result showed that 2,4-D could induce a parthenocarpic-like growth. The rates of fresh weight, dry weight , water content, and sugar accumulation in 2,4-D treated kernels were faster than that of pollinated kernels during early developing stage (1~5 days after treatment). After 5 days of development, the physiological processes were decreased. In morphological observation, the auxin could promote pericarp and nucellus growth. However, no endosperm organ and the accumulation of starch and storage protein (zein) could be detected in 2,4-D induced parthenocarpic kernels. This suggested that auxin can promote a fast growth of unpollinated maize caryopsis during early developing stage, but it could not enable the caryopsis to develop into a functional seed. With 2-D gel analysis, several proteins were up-regulated by 2,4-D and were identified, including: germin-like protein 1, calmodulin, glycine-rich RNA binding protein 1, and profilin. Germin-like protein 1 expressed in kernels of 3 days after 2,4-D treatment and in kernels of 3 days after pollination (DAP). The other proteins expressed later in kernels of 5 days after 2,4-D treatment and in kernels of 5 DAP. The function of these proteins may be correlated with the auxin induced signal transduction and rapid tissue growth. In pollinated kernels several glycolytic related enzymes were identified from kernels at 12 DAP, for example, G 3-P dehydrogenase and fructose bisphosphate aldolase. These proteins have been suggested to be involved in energy supply for the growing cell activity. Significant expressions of chitinase A and 22K antifungal proteins were interestingly observed in unpollinated and degenerating kernels, which might be related to the protection of infection during senescence of caryopsis without pollination. In this study, 40 proteins in kernel of early developing stage were identified. The proteome profiles will facilitate future studies addressing the effects of genetic and environmental factors on the development and quality of maize kernels.