擬南芥轉殖株百合LLA23蛋白參與離層酸和葡萄糖訊息傳遞及耐旱抗鹽特性

Abstract

LLA23為鐵砲百合（Lilium longiflorum Thunb. cv. Snow Queen）花粉專一性蛋白質，在花粉發育成熟的乾燥時期會大量累積。LLA23屬於ABA-, stress-, and ripening-induced (ASR)蛋白質的一員。為了了解百合ASR蛋白質在植物體內扮演的角色，本研究利用35S啟動子將LLA23轉殖入擬南芥，並以北方及西方墨跡法加以確認。雖然35S::LLA23轉殖株不論是外表型態或是開花時間，皆與野生型無異，但是種子萌發試驗發現35S::LLA23兩轉殖株種子的萌發對離層酸較不敏感。種子休眠試驗發現35S::LLA23兩轉殖株種子與abi4-1相似。轉殖株種子亦展現對高鹽及高滲透壓的抗性，具有較高的萌發率。以上兩轉殖株對離層酸的不敏感特性暗示著百合ASR蛋白質在離層酸之訊息傳遞路徑上可能扮演了調控的角色。即時定量聚合酵素鏈鎖反應的結果顯示，不論有無離層酸處理，百合LLA23蛋白質確實在轉殖植物體內影響了逆境/離層酸反應相關基因的表現。35S::LLA23轉殖植株在乾旱或高鹽處理下較野生型有較高的抗性。缺水12天的野生型植株大部分呈現萎凋，葉片氣孔多為關閉，而兩轉殖株有半數仍然直立，葉片生長正常且氣孔多為開啟。缺水12天的野生型植株葉片離層酸含量遽增近10倍，然而兩轉殖株的離層酸含量沒有太大的變化，說明了轉殖株葉片的氣孔仍然保持敞開的原因。進一步測量滲透勢值，轉殖株葉的滲透勢值與野生型相似，表示轉殖株在乾旱12天下尚不缺水。乾旱過程中葉片水份喪失的比例較野生型慢，而其氣孔卻多為開啟的狀態，顯示百合LLA23蛋白質在葉片中可能扮演保水，不讓水分流失的角色。若再繼續缺水4天，兩轉殖株滲透勢值達到-2.55及-2.98 MPa，呈現植物體內極度缺水，因此轉殖株開始萎凋，葉片氣孔多轉為關閉。葉片蒸散試驗顯示，轉殖株葉片水份喪失程度與野生型相同，但轉殖株的葉片氣孔卻多為打開的狀態，同樣顯示百合LLA23蛋白質在葉內不使水分流失的功能。 種子萌發試驗發現，35S::LLA23轉殖株種子的萌發和後萌發時期小苗的生長對高濃度葡萄糖及甘露糖皆較不敏感。利用六碳糖激酶之競爭型抑制劑，甘露庚酮糖處理下，不論野生型或轉殖株種子萌發受甘露糖抑制的現象，在加入甘露庚酮糖後皆會消失，證實百合ASR蛋白質參與甘露糖經由六碳糖激酶中介的訊息傳遞路徑。低濃度 (10 mM)葡萄糖可解除甘露糖之抑制現象，而35S::LLA23轉殖株不論在種子萌發或後萌發時期之根系生長，對葡萄糖解除甘露糖之抑制現象具較高的敏感性。即時定量聚合酵素鏈鎖反應結果顯示，百合LLA23蛋白質在高糖處理下，確實影響了轉殖植物體內一些逆境相關基因的表現。顯示百合LLA23蛋白質可能參與葡萄糖之訊息傳遞。 百合LLA23基因含有一個插入子，啟動子序列中有許多與離層酸相關的保留序列。百合LLA23蛋白質具有轉錄活化的區段可能在氨基酸17~57之間。利用染色質免疫沉澱分析方式可確定百合LLA23蛋白質與染色質進行結合。蛋白質的結晶試驗仍在進行中。

Abstract

LLA23, a member of abscisic acid-, stress-, and ripening-induced (ASR) family protein previously isolated from lily (Lilium longiflorum) pollen was abundantly accumulated upon desiccation during anther/pollen development. To provide evidence on the biological role of LLA23 proteins against drought, we used an overexpression approach in Arabidopsis (Arabidopsis thaliana). Northern and Western analyses confirmed the presence of transgene of LLA23 in transgenic plants. Constitutive expression of LLA23 under the cauliflower mosaic virus 35S promoter confered reduced sensitivity to ABA in transgenic seeds and consequently, a reduced degree of seed dormancy which was very similar to that of the abi4-1 mutant. 35S::LLA23 transgenic seeds were able to germinate under unfavorable conditions, such as inhibitory concentrations of mannitol and NaCl. At the molecular level, altered expression of ABA/stress-regulated genes was observed. Thus, our results provide strong in vivo evidence that LLA23 mediates stress-responsive ABA signaling. 35S::LLA23 transgenic plants improved drought and salt resistance. When soil in pots was withheld water for 12 d, most of the wild-type plants became wilted and stomata remained closed whereas about 50% of the two 35S::LLA23 plants kept upstanding and stomata remained open. The level of ABA content in wild-type leaves dramatically increased 10 folds after 12 d of drought stresses when compared with the unstressed leaves. It explains the reason why 35S::LLA23 stomata remained open. Further, the osmotic potential in 35S::LLA23 leaves did not appreciably change upon drought stress for 12 d indicating that the transgenic plants did not sense drought stress. The water loss in transgenic plants was slower than that in wild-type plants at meantime transgenic stomata remained open, reflecting that the LLA23 protein has the water holding ability in transgenic plants. If these plants were continued to withhold water for additional 4 d, transgenic plants began to wilt and their ABA levels in leaves markedly increased. Moreover, most 35S::LLA23 stomata became closed and osmotic potential increased to -2.55 and -2.98 MPa. In vegetative tissues, the transpiration rate in transgenic leaves under drought stress was similar to that in wild-type plants, but most of the 35S::LLA23 stomata remained open also suggesting of a protective role of LLA23 proteins. Constitutive expression of LLA23 in Arabidopsis confers reduced sensitivity to mannose and high concentration of glucose in transgenic seed germination and post-germination. Under the treatment of mannoheptulose, a specific hexokinase inhibitor, both the wild-type and transgenic plants restored mannose-repressed seed germination. It suggests that LLA23 involving signaling is by way of hexokinase-mediated pathway. Low concentration of glucose (10 mM) can relieve mannose-repressed seed germination while 35S::LLA23 seeds and seedings exhibit hypersensitivity on the relief of the mannose-repressed germination and seedling development. At the molecular level, altered expression of stress-regulated genes was observed when 35S::LLA23 seedling was treated with high concentration of glucose. It reinforces the possibility that LLA23 mediates glucose signaling. The LLA23 gene contains a single intron and the identified promoter of LLA23 has potential regulatory elements in response to abscisic scid. The LLA23 has a transactivation domain as a fragment from amino acid 17 to 57 of the sequence. Chromatin immunoprecipitation assay revealed that LLA23 can bind DNA. The crystallization of LLA23 proteins is in progress.