Plant desiccation tolerance: from physiology of seeds to the molecular mechanism of vegetative tissues

細胞膜磷脂質的組成在種子適應乾燥逆境扮演重要角色。因此，我們分析了許多種乾儲型和異儲型種子在胚軸的磷脂組成。結果顯示，這兩類種子的胚軸在phosphatidyl ethanolamine (PE)和phosphatidylcholine (PC)的比值有明顯的重疊。在磷脂的脂肪酸飽和度上，乾儲型種子飽和脂肪酸佔的比例(16% to 27%)明顯低於異儲型種子(32% to 36%)，這個差異也反映在細胞膜的相變溫度上，而兩類種子在非飽和脂肪酸的比例上則無顯著差異。在以萌發的大豆種子模擬異儲型種子的實驗中，PE和PC的比值先增後減。萌芽後失去耐乾燥能力的胚軸在磷脂脂肪酸飽和度上明顯增加。這個結果與乾、異儲型種子的比較結果相符。然而，飽和度的增加並未反映在萃取出來的磷脂的相變溫度上，這可能是linolenic acids所造成的影響。總之，不耐乾燥的異儲型種子和萌發的大豆胚軸具有較高的磷脂飽和度，較高含量的linolenic acids與較高的細胞膜相變溫度。年松是最原始的維管束植物之一，它可以在乾燥下存活，當重新獲得水分時恢復生機。為了了解萬年松忍受乾燥的特性，我們利用氣相層析儀分析乾燥前後萬年松在可溶性醣和磷脂飽和度上的差異。此外，我們也分析了萬年松在乾燥過程蛋白質、超氧歧化酵素活性、基因表現和ABA含量的變化。結果顯示海藻糖是萬年松主要的可溶性醣(以乾重計算，每克萬年松含量超過130毫克)，其含量在乾燥前後無明顯差異。乾燥前、後的植物組織在細胞膜磷脂上皆維持低的脂肪酸飽和度(0.31)。乾燥過程中可以看到新的蛋白質被合成與新的超氧歧化酵素活性被活化。此外，乾燥過程ABA含量增加三倍，ABA訊息傳遞和細胞保護相關基因被誘導表現，光合作用相關基因的表現則被抑制。由這些生化和分子生物上的證據顯示持續性的高雙醣含量和低磷脂飽和度與乾燥誘導ABA增加和基因表現皆參與了萬年松忍受乾燥的機制。們利用differential display的方式，在萬年松篩選到一個受缺水誘導表現的基因，StCaLB。此基因的產物是一個calcium dependent lipid binding蛋白。為了進一步研究此基因的功能，我們分析了這個基因在不同非生物性逆境下的表現和protein domain的預測，並將此基因的cDNA轉入阿拉伯芥中持續性的表達。結果顯示，StCaLB的表現會受到鹽、30% PEG、熱和ABA的誘導但不受低溫處理影響。蛋白質domain的預測顯示，StCaLB在N端有兩個transmembrane domain，在蛋白質中段有一個C2 domain。另外我們也得到StCaLB持續性表達的阿拉伯芥轉植株。然而，StCaLB的功能仍有賴更進一步的生化與遺傳方面的分析。Membrane phospholipids play an important role in acclimation of seeds to desiccation stresses. The compositions of phospholipids in embryonic axes of several recalcitrant seeds and orthodox seeds were studied. The ratio of phosphatidyl ethanolamine to phosphatidylcholine in embryonic axes of recalcitrant seeds and orthodox seeds was overlapping in the range. Percentage of saturated fatty acids of phospholipid (stearic acid, C18:0, and palmitic acid, C16:0) was significantly different between orthodox seeds (in the range of 16% to 27%) and recalcitrant seeds (32% to 36%), and this difference also reflected in the lower phase transition temperature of orthodox seeds. The proportion of unsaturated fatty acids (C18:2 and C18:3) of phospholipids was overlapping in both seed types. Using imbibed soybean seedlings as a model system for recalcitrance, the ratio of phosphatidyl ethanolamine over phosphatidylcholine of embryonic axes increased in early imbibition and decreased later on. The percentage of saturated fatty acids in phospholipids of soybean seedlings excluding cotyledon was significantly higher than that in dry embryonic axis. This agrees with the results from the comparison of recalcitrant and orthodox seeds. Increases in saturated fatty acids were not correlated with lower transition temperature of extracted phospholipids of seedling indicating linolenic acids (C18:3) might play a role on Tm value. In conclusion, both recalcitrant seeds and imbibed seedlings while sharing recalcitrance, exhibited a similar trend in fatty acid saturation and higher percentage of linolenic acid in phospholipids, and higher phase transition temperature when compared with orthodox seeds and dry embryonic axes. elaginella tamariscina, one of the most primitive vascular plants, can remain alive in a desiccated state and resurrect when water becomes available. To evaluate the nature of desiccation tolerance in this plant, we compared the composition of soluble sugars and saturation ratios of phospholipids (PLs) between hydrated and desiccated tissues of S. tamariscina using gas chromatography (GC). In this study, differences in protein dynamics, superoxide dismutase (SOD) activities, gene expressions and abscisic acid (ABA) contents were also analyzed during dehydration. The results revealed that trehalose (at > 130 mg g-1 dry weight) was the major soluble sugar, and low saturated fatty acid contents in PL (0.31) was maintained in both hydrated and desiccated tissues. Novel proteins and an inducible CuZnSOD activity were detected during dehydration. In addition, the ABA content of S. tamariscina increased 3 folds, and genes involved in ABA signaling and cellular protection were upregulated while photosystem-related genes were downregulated during dehydration. The biochemical and molecular findings suggest that both constitutive and inducible protective molecules contribute to desiccation tolerance of S. tamariscina.o study the function of StCaLB putatively encoding a calcium dependent lipid binding protein screened from differential display of Selaginella tamariscina during dehydration, the genomic sequence, putative protein domain and the response of this gene to different abiotic stresses were analyzed. Also, the transgenic plants of Arabidopsis that over-expressing StCaLB were generated. The results indicated the expression of StCaLB is induced by NaCl, 30% PEG, heat and abscisic acid, but not by cold treatment. The StCaLB is predicted to contain two N-terminal transmembrane domains and a central C2 domain. The StCaLB over-expressing Arabidopsis were obtained. However, further biochemistry and genetic research still need to be evaluated for the function of this gene.Table of Contentsagebbreviations…………………………………………………...…………………….1hapter 1: Comparison of phospholipids and their fatty acids in recalcitrant and orthodox seeds…………………………………………………………………………3bstract in Chinese…………………………………………………………………..3bstract in English……………………………………………...……………………4ntroduction…………………………………………………………………………..6aterials and Methods………………………………………………………………8lant materials………………………………………………………………………..8oybean seed germination and treatment…………………………………………….9ipid extraction and analysis………………………………………………………..10hospholipid analysis……………………………………………………………….10atty acid analysis..…………………………………………………………………11ourier transform infrared spectroscopy (FTIR)..…………………………………..12esults……………………………………………………….……………………….13omposition of PL classes in various seeds………………………………………...14eight percentage of fatty acids in PL of recalcitrant and orthodox seed….............14m for embryonic tissue of recalcitrant seed and orthodox seed……………...…….15hanges of composition of PL and their fatty acids of soybean seeds imbibed for up to 32 h at 25 oC………………………………..…………………………………….15m for imbibed seedlings and extracted PLs of soybean……………………………16iscussion……………………………………………………………………………16igures and tables…………………………………………………………………...22hapter 2: Constitutive components and induced gene expressions are involved in the desiccation tolerance of Selaginella tamariscina…………………...……………30bstract in Chinese…………………………………………………………………30bstract in English………………….………………………………………………31ntroduction…………………………………………………………………………32aterials and Methods……………………………………………………………..35lant materials………………………………………………………………………35ater content determination……………………………………….……………….36on leakage analysis…………………….…………………………………………..36xtraction and analysis of phospholipids (PLs) and soluble sugars….…………….37xtraction, purification, and quantification of ABA……………………….……….38rotein extraction and two dimensional protein analysis………….………………..40OD activity analyses………………………………………………………………41ehydration activated cDNA clone isolation and DNA sequence analysis….….….42election of differentially expressed genes during dehydration of S. tamariscina....42orthern blot analysis of gene expression in dehydration- and ABA-treated S. tamariscina…………………………………………………………………….…..44esults………………………………………………………….……………………45 Resurrection phenomenon of S. tamariscina……………………………………….45ow saturation ratio of fatty acids in PLs was maintained during dehydration……46rehalose was the main soluble sugar detected in S. tamariscina…………….……47BA increased in dehydrated S. tamariscina……………………………………….47ovel proteins were synthesized in desiccated S. tamariscina………………..……48 uZn-SOD activities were activated during dehydration in S. tamariscina………..48ighteen unique genes were upregulated during dehydration in S. tamariscina…...49 calcium dependent lipid binding protein gene and a hydrolase gene were specifically expressed during dehydration…………………….……………………49wenty-two unique genes were differentially expressed between hydrated and dehydrated S. tamariscina……………….……………………………………….…49 BA-dependent gene expressions were revealed in S. tamariscina…….……….…51 iscussion……………………………………………………………………………52he membrane system of S. tamariscina was well protected during desiccation and recruit of water…..………………………………………………………………….52ow saturation of PLs in membranes was found in S. tamariscina……………...…53ccumulation of a high level of disaccharide is a characteristic of lower vascular plants………………………………………………………………………………..54n inducible protective mechanism is involved in acquisition of desiccation tolerance in S. tamariscina………………………………………………………….56igures and tables………………..………………………………………………….62hapter 3: A gene encodes a putative calcium dependent lipid binding protein was response to diverse abiotic stresses in Selaginella tamariscina…………………..….78bstract in Chinese…………………………………………………………………78bstract in English………………….………………………………………………79ntroduction…………………………………………………………………………80aterials and Methods……………………………………………………………..80lant materials………………………………………………………………………80orthern blot analysis of gene expression under different abiotic stresses…….…..81ene construction and plant transformation………………………………………..81esults………………………………………………………….……………………82 The cloning and expression pattern of the putative calcium dependent lipid binding protein gene of S. tamariscina…………………………………….……..………….82tCaLB overexpressed in Arabidopsis….……………………….………………..…83 iscussion……………………………………………………………………………83igures and tables………………..………………………………………………….85eferences………………………………………………………………………...…89ppendix: Nucleotide sequence of differentially expressed genes identified in S. tamariscina…………………………………………………………………....….....10