臺灣博碩士論文加值系統

香蘭（Pandanus amaryllifolius Roxb.）生長於東南亞為露兜樹種中唯一葉片帶有芳香氣味的植物。香味的來源主要是2-acetyl-1-pyrroline（2-AP）。2-AP化學結構中pyrroline的氮原子已證實來自於脯胺酸。因此脯胺酸的代謝反應，可能在2-AP的生合成扮演重要角色。另一方面，亦有報告指出，由二羥基丙酮磷酸 (DHAP) 降解生成的methylglyoxal (MG) 與2-AP的生合成有關係。本實驗擬利用照光及避光處理來探討香蘭中香氣化合物2-AP生合成之機制。在實驗初期發現經過日照後之香蘭葉片所含2-AP含量較避光高。以RT-PCR方式來分析比較照光及避光下，在香蘭中2-AP合成關鍵基因的表現量並測定葉片中的MG、P5C和2AP的含量。結果顯示在合成MG之相關基因中，照光下GapA和GapB之mRNA表現量最高而GapC的表現量在照光下則較低。因為GapA及GapB存在於葉綠體中為合成GAP的關鍵酵素，而GapC存在細胞質內負責代謝GAP，由此結果可推測GAP蓄積而增加形成MG的機會。而經過分析之後所得之MG、P5C含量皆為照光多於避光，顯示MG與P5C之含量皆與2-AP的濃度成正相關。此外，由香蘭中選殖出與MG生合成相關之關鍵酵素，包含GAPDH之次單位GapA、GapB 和GapC。經過蛋白質表現重組蛋白質GapA、GapB和GapC之分子量分別為43.3KDa、34.2KDa和36.7KDa。經Ni2+親和性管柱純化後之重組蛋白GapC測得其比活性為5.11 U/mg。

Pandanus amaryllifolius Roxb. (Pandan) grows in Southeast Asia and is the only reported pandanus species with scented leaves. The aroma source is mainly 2-acetyl-1-pyrroline. The nitrogen source of pyrroline of 2-AP structure was proline. Proline metabolism may play a crucial role in 2-AP formation. On the other hand, methylglyoxal (MG) may derive from dihydroxyacetone phosphate (DHAP) was proposed to be responsible for 2-AP formation. This study probed into the formation mechanism of 2-AP in pandan under light or dark. In the preliminary experiment, the level of 2-AP was higher in pandan leave under light than under dark. The mRNA expression of the 2-AP synthesis related genes were analyzed with gene-specific primers by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) in pandan under light and dark. MG, P5C and 2-AP contents in pandan leaves under either light or dark were examined. The levels of expression of GapC mRNA under light were the lowest. GapC exists in the cytosol and is responsible for the metabolism of GAP. The lower activity of GapC in lighted pandan leave may be attributed to the accumulation of MG. The levels of both P5C and MG were higher in pandan leave under light than that in dark were found to be parallel to the 2-AP level. A formation mechanism of 2-AP from interation between P5C and MG was proposed. In addition, the key enzymes from pandan for MG biosynthesis, including the subunit A (GapA), B (GapB) and C (GapC) of GAPDH were cloned. Overexpression of GAPDH of the recombinant proteins exhibit a molecular mass of 43.3KDa, 34.3KDa and 36.7KDa, respectively. Specific activities of GapC of the recombinant proteins purified by Ni2+ affinity column were determined as 5.11 U/mg.

中文摘要 I
Abstract III
誌謝 V
目錄 VI
圖表目錄 XI
第1章 前言 1
第2章 文獻回顧 3
2.1 香氣成分 2-acetyl-1-pyrroline 之簡介 3
2.1.1 2-acetyl-1-pyrroline化學合成機制之探討 3
2.1.2生物體中2-acetyl-1-pyrroline生合成之探討 7
2.2 生物體內脯胺酸生合成之調控機制 9
2.2.1 P5C生合成路徑 9
2.2.2脯胺酸的生理功能 10
2.2.3 P5C合成酶 (△1-Pyrroline -5-Carboxylate Synthetase,
P5CS) 之特性及相關研究 11
2.2.4 脯胺酸去氫酶 (Proline dehydrogenase, PDH) 之特性
及相關研究 13
2.2.5 鳥胺酸轉胺酶 (Ornithine-δ-aminotransferase, OAT)
之特性及相關研究 13
2.2.6 P5C之毒性與解毒機制 14
2.3 Methylglyoxal (MG) 之形成與相關研究 14
2.3.1 Methylglyoxal之介紹與形成機制 14
2.3.2 Methylglyoxal對生物體的毒性 17
2.3.3 MG之解毒機制 18
2.3.4 Methylglyoxal生合成相關基因之介紹 19
2.4 研究目的 23
第3章 材料與方法 24
3.1 物種來源 24
3.2 載體 24
3.3 大腸桿菌菌種 24
3.4 儀器設備 24
3.5 香蘭葉片總量RNA之萃取 26
3.5.1 藥品 26
3.5.2 藥品配製 26
3.5.3 總量RNA萃取步驟 27
3.5.4 RNA電泳(RNA electrophoresis) 27
3.5.5 核糖核酸濃度的定量 27
3.6 反轉錄作用 (reverse transcription) 28
3.7 退化性引子 (degenerate primer) 之設計 28
3.8 聚合酶連鎖反應 (polymerase chain reaction, PCR) 29
3.9 DNA 電泳(DNA electrophoresis) 29
3.9.1藥品配製 29
3.9.2 DNA電泳流程 29
3.10 TA Cloning接合反應(Ligation) 30
3.11 勝任細胞之製備(E. coli XL1-Blue competent cell) 30
3.12 選殖載體轉型進入勝任細胞(transformation) 30
3.13 藍白篩選 31
3.14 以PCR方式確定是否含有所選殖片段 31
3.15 大量增殖含有選殖片段之菌落 31
3.16 小量質體之萃取 31
3.17 定序(sequencing) 32
3.18 cDNA尾端快速增殖法(rapid amplification of cDNA ends,
RACE) 32
3.18.1設計Rapid Amplification of cDNA Ends (RACE)專用
引子 32
3.18.2 poly (A)+ RNA的純化 33
3.18.3合成RACE cDNA 33
3.18.4 RACE PCR 34
3.19 全長DNA之選殖 34
3.20 序列分析 34
3.21 GapA、GapB及GapC表現質體之建構 35
3.21.1 以具有校正功能之聚合酶進行PCR 35
3.21.2 限制酵素處理PCR產物及pET21b載體 35
3.21.3 接合作用 35
3.21.4 轉型 36
3.21.5 選殖 36
3.22 菌種保存 36
3.23 重組蛋白質之表現 36
3.23.1勝任細胞BL21(DE3)之製備 36
3.23.2表現質體轉型入大腸桿菌BL21(DE3) 37
3.23.3大腸桿菌中表現目標蛋白 37
3.24 聚丙烯醯胺膠體電泳(Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis, SDS-PAGE) 37
3.24.1藥品配製 37
3.24.2聚丙烯醯胺膠體之製備 38
3.24.3操作流程 38
3.25 重組蛋白質之純化 39
3.25.1藥品配製 39
3.25.2管柱清洗 39
3.25.3純化步驟 39
3.26 蛋白質之定量方法 40
3.27 GapC之活性分析 40
3.28 香蘭葉片之酵素活性分析 40
3.28.1總蛋白質之萃取 40
3.28.2 GapA及GapB活性分析方法 41
3.28.3 GapC活性分析方法 41
3.28.4 TPI活性分析方法 41
3.28.5 FBA活性分析方法 42
3.28.6 GK活性分析方法 42
3.28.7 OAT活性分析方法 42
3.28.8 P5CS活性分析方法 43
3.28.9 PDH活性分析方法 43
3.29 RT-PCR分析照光及避光處理香蘭葉片之mRNA表現量 43
3.30 2-AP之定量分析 44
3.31 P5C呈色反應 44
3.32 MG之定量分析 44
3.32.1萃取方法 45
3.32.2定量分析 45
第4章 結果 46
4.1 2-AP之香氣鑑定 46
4.2 香蘭總量RNA之萃取以及cDNA之合成 46
4.3 RT-PCR之分析 46
4.3.1 MG生合成相關基因之mRNA表現量 46
4.3.2 P5C生合成相關基因之mRNA表現量 47
4.3.3 MG代謝相關基因之mRNA表現量 47
4.4 香蘭葉片總蛋白質活性之分析 48
4.5 香蘭葉片中P5C之呈色分析 48
4.6 香蘭葉片中MG之分析 49
4.7 香蘭中GapA、GapB及GapC基因之選殖 49
4.7.1 GapA基因之選殖 49
4.7.2 GapB基因之選殖 49
4.7.3 GapC基因之選殖 50
4.8 香蘭中GapA、GapB及GapC基因及胺基酸之序列比對 50
4.8.1 GapA之序列比對 50
4.8.2 GapB基因之序列比對 51
4.8.3 GapC基因之序列比對 51
4.9 表現質體之建構 51
4.9.1 GapA表現質體建構 51
4.9.2 GapB表現質體建構 52
4.9.3 GapC表現質體建構 52
4.10香蘭GapA、GapB、及GapC於E. coli中之表現 53
4.10.1 GapA重組蛋白之表現 53
4.10.2 GapB重組蛋白之表現 53
4.10.3 GapC重組蛋白之表現 53
4.11 GapC重組蛋白純化與活性分析 54
第5章 討論 89
第6章 結論 92
參考文獻 93
作者簡介 107
圖表目錄
圖1、 2-acetyl-1-pyrroline的結構 3
圖2、 Proline和2-oxopropanal作用形成ATHP、1-pyrroline和hydroxy-2-propanone 4
圖3、 脯胺酸及pyruvaldehyde 反應形成麵包香氣化合物 5
圖4、 鳥胺酸產生1-pyrroline的可能反應機制 6
圖5、 1-pyrroline與methylglyoxal反應形成2-acetyl-1-pyrroline 7
圖6、 2-acetyl-1-pyrroline之可能生合成途徑 9
圖7、 麩胺酸、鳥胺酸和脯胺酸生合成路徑 10
圖8、 麩胺酸、鳥胺酸及脯胺酸之間代謝圖 11
圖9、 P5CS和PDH在乾燥與復水的環境條件的調節作用 12
圖10、經三碳醣磷酸異構酶催化產生methylglyoxal之反應 15
圖11、MG的生合成路徑 16
圖12、氧化損傷後所產生之羰基化合物之結構及其細胞毒性 17
圖13、MG代謝路徑 19
圖14、卡爾文循環 21
圖15、醣解作用 22
圖16、GC-MASS分析香蘭中2-AP 55
圖17、香蘭葉片總量RNA萃取之電泳分析圖 56
圖18、GapA、GapB、GapC、FBA及TPI 之RT-PCR電泳分析圖 58
圖19、P5CS及OAT之RT-PCR電泳分析圖 59
圖20、AR、BAD及GST之RT-PCR電泳分析圖 60
圖21、照光及避光處理期間香蘭中P5C之分析 63
圖22、HPLC分析香蘭中MG。 64
圖23、選殖香蘭中基因cDNA轉譯甘油醛三磷酸去氫酶A次單位(GapA)之電泳圖 70
圖24、選殖香蘭中基因cDNA轉譯甘油醛三磷酸去氫酶B次單位(GapB)之電泳圖 71
圖25、選殖香蘭中基因cDNA轉譯甘油醛三磷酸去氫酶C次單位(GapC)之電泳圖 72
圖26、香蘭GapA cDNA之核苷酸與轉譯的胺基酸序列 76
圖27、香蘭之GapB cDNA之核苷酸與轉譯的胺基酸序列 77
圖28、香蘭之GapC cDNA之核苷酸與轉譯的胺基酸序列 78
圖29、香蘭GapA與其他植物胺基酸序列比對分析 79
圖30、香蘭GapB與其他植物胺基酸序列比對分析 80
圖31、香蘭GapC與其他植物胺基酸序列比對分析 81
圖32、(A) pET-21之限制圖譜 (B) pET21-GapA經限制酶BamHI和
XhoI作用之電泳分析圖 82
圖33、(A) pET-21之限制圖譜 (B) pET21-GapB經限制酶EcoRI和
XhoI作用之電泳分析圖 83
圖34、(A) pET-21之限制圖譜 (B) pET21-GapC經限制酶BamHI和
XhoI作用之電泳分析圖 84
圖35、重組蛋白GapA之SDS-PAGE分析 85
圖36、重組蛋白GapB之SDS-PAGE分析 86
圖37、重組蛋白GapC純化之SDS-PAGE分析 87

表1、 MG對EAC細胞醣解酵素之影響 22
表2、 RT-PCR分析之引子 57
表3、 香蘭中MG生合成相關酵素之酵素活性 61
表4、 香蘭中P5C生合成相關酵素之酵素活性 62
表5、 照光及避光香蘭葉中MG之濃度分析 65
表6、 選殖片段基因之退化性引子 66
表7、 選殖cDNA末端序列之引子(RACE) 67
表8、 選殖全長cDNA之引子 68
表9、 於大腸桿菌表現之引子 69
表10、GapA基因序列與其它物種之相似度比較 73
表11、GapB基因序列與其它物種之相似度比較 74
表12、GapC基因序列與其它物種之相似度比較 75
表13、從E. coli BL21 (DE3)純化出香蘭GapC之純化表 88

莊惠夙，2005。香米主要香氣成分2-Acetyl-1-Pyrroline生物合成之相關基因的選殖與表現分析。國立屏東科技大學食品科學研究所碩士論文。

陳建凱，2005。毛豆中2-acetyl-1-pyrroline生物合成相關基因之選殖與表現分析。國立屏東科技大學食品科學研究所碩士論文。

華東師範大學網路教育學院，2005。光合作用的機制(難點，重點)。卡爾文循環。2005年9月29日，取自：http://rts.dec.ecnu.edu.cn/zsb/zsw/zsw08/zsw083/zsw08303/zsw083030.htm

Alia-Saradhi PP. 1993. Supression in mitochondrial electron transport is the prime cause behind stress induced praline accumulation. Biochemical and biophysical research communications 193: 54-8.

Andrews DL, MacAlpine DM, Johnson JR, Kelley PM, Cobb BG, Drew MC. 1994. Differential induction of mRNAs for the glycolytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings. Plant Physiology 106: 1575-82.

Baalmann E, Backhausen JE, Rak C, Vetter S, Scheibe R. 1995. Reductive modification and Nonreductive activation of purified spinach chloroplast NADP-dependent glyceraldehydes-3-phosphate dehydrogenase. Archives of Biochemistry and Biophysics 324: 201-8.

Beisswenger PJ, Howell SK, Smith K, Szwergold BS. 2003. Glyceraldehyde-3-phosphate dehydrogenase activity as an independent modifier of methylglyoxal levels in diabetes. Biochimica et Biophysica Acta 1637: 98-106.

Bonner CA, William DS, Aldrich HC, Jensen RA. 1996. Antagonism by L-glutamine of toxicity and growth inhibition caused by other amino acids in suspension cultures of Nicotiana silvestris. Plant Science 113: 43-58.

Brahmachary RL, Ghosh M. 2000. Baginal pheromone and other unusual compounds in mung bean aroma. Current Science 78: 1410-7.

Brinkmann H, Cerff R, Salomon M, Soll J. 1989. Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehydes-3-phosphate dehydrogenase from pea and spinach. Plant Molecular Biology 13: 81-94.

Buchi G, Wuest H. 1971. Synthesis of 2-acetyl-1,4,5,6-tetrahydropyridine, a constituent of bread aroma. Journal of Organometallic Chemistry 36: 609-10.

Buttery RG, Ling LC. 1982. 2-acetyl-1-pyrroline: An important aroma component of cooked rice. Chemical Industry (London) 958-9.

Buttery RG, Juliano BO, Ling LC, Turnbaugh JG. 1983. Cooked Rice Aroma and 2-Acetyl-1-pyrroline. Journal Agricultural and Food Chemistry 31: 823-8.

Buttery RG, Juliano BO, Ling LC. 1983. Identification of rice aroma compounds 2-acetyl-1-pyrroline in Pandan leves. Chemical Industry 478.

Buttery RG, Ling LC, Juliano BO. 1985. 2-acetyl-1-pyrroline and its use for flavoring foods. US patent. 452: 28-38.

Buttery RG, Ling LC, Mon TR. 1986. Quantitative analysis of 2-acetyl-1-pyrroline in rice. Journal Agricultural and Food Chemistry 34: 112-4.

Buttery RG, Turnbaugh JG, Ling LC. 1988. Contribution of volatiles to rice aroma. Journal Agricultural and Food Chemistry 36: 1006-9.

Cerff R. 1979. Quaternary structure of higher plant glyceraldehydes-3-phosphate dehydrogenase. European Journal of Biochemistry 94: 243-7.

Cerff R, Chambers SE. 1979. Subunit structure of higher plant glyceraldehyde-3-phosphate dehydrogenase (EC1.2.1.12 and EC1.2.1.13). Journal Biological Chemistry 254: 6094-8.

Chang YC, Lee TM. 1999. High temperature-induced free proline accumulation in Gracilaria tenuistipitata (Rhodophyta). Botanical Bulletin Academia Sinica 40: 289-94.

Cordeiro C, Freire P. 1995. Methylglyoxal assay in cells as 2-methylquinoxaline using 1,2-diaminobenzene as derivatizing reagent. Analytical Biochemistry 234: 221-4.

Degols G. 1987. Functional analysis of the regulatory region adjacent to the carB gene of Saccharomyces cerevisiae. European Journal of Biochemistry 169: 193-200.

Delauney AJ, Hu CA, Kishor PBK, Verma PS. 1993. Cloning of ornithine-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. Journal Biological Chemistry 268: 18673-8.

Delauney AJ, Verma DPS. 1993. Proline biosynthesis and osmoregulation in plants. The Plant Journal 4: 215-23.

Deswal R, Chakravarty TN, Sopory SK. 1993. The glyoxalase system in higher plants: regulation in growth and differentiation. Biochemical Society Transactions 21: 527-30.

Deuschle K, Funck D, Hellmann H, Däschner K, Binder S, Frommer WB. 2001. A nuclear gene encoding mitochondrial △1 –pyrroline-5- carboxylate dehydrogenase and its potential role in protection from proline toxicity. The Plant Journal 27: 345-55.

Faggin P, Bassi AM, Finollo R, Brambilla G. 1985. Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal. Mutation Research 144: 189-91.

Fleming GA, Granger A, Rogers QR, Prosser M, Ford DB, Phang JM. 1989. Fluctuations in plasma pyrroline-5-carboxylate concen-trations during feeding and fasting. Journal of Clinical Endocrinology and Metabolism 69: 448-452.

Fothergill-Gilmore LA, Michel PA. 1993. Evolution of glycolysis. Progress in Biophysics and Molecular Biology 59: 105-235.

Fushimi T, Masuda R. 2001. 2-acetyl-1-pyrroline concentration of the aromatic vegetable soybean “Dadacha-Mame”. Conference Proceedings, Washington, USA. p 39.

Fujita M, Nanjo T, Seki M, Kato T, Tabata S, Shinozaki K. 2003. Toxicity of free proline revealed in an Arabiposis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiology 44: 541-8.

Garden R, Rapoport G, Débarbouillé M. 1995. Expression of the roc DEF operon involved in the arginine catabolism in Bacillus subtilis. Journal of Molecular Biology 249: 843-56.

Halder J, Ray M, Ray S. 1993. Inhibition of glycolysis and mitochondrial respiration of Ehrlich ascites carcinoma cells by methylglyoxal. International Journal of Cancer 54: 443-9.

Hayashi T, Shibamoto T. 1985. Analysis of methylglyoxal in foods and beverages. Journal of Agricultural and Food Chemistry 33: 1090-3.

Heimberg H, Boyen A, Crabeel M, Glandsdorff N. 1990. Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferase: evolutionary relationship with ornithine aminotransferase. Gene 90: 69-78.

Hellmann H, Funck D, Rentsch D, Frommer WB. 2000. Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application. Plant Physiology 123: 779-90.

Henze K, Schnarrenberger C, Kellermann J, Martin W. 1994. Chloroplast and cytosolic triosephosphate isomerase from spinach: purification, microsequencing and cDNA cloning of the chloroplast enzyme. Plant Molecular Biology 26: 1961-73.

Heyser JW, De Bruin D, Kincaid ML, Johnson RY, Rodriguez MM, Robinson NJ. 1989. Inhibition of NaCl-induced proline biosynthesis by exogenous proline in halophilic Distichlis spicata suspension cultures. Journal of Experimental Botany 40: 225-232.

Hofmann T, Schieberle P. 1998. 2-oxopropanal, hydroxy-2-propanone, and 1-pyrroline-important intermediates in the generation of the roast-smelling food flavor compounds 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine. Journal of Agricultural and Food Chemistry 46: 2270-7.

Hodge JE, Mills FD, Fisher BE. 1972. Compounds derived from browned flavors derived from suger-amine reaction. Cereal Science Today 17: 34-40.

Hu CA, Delauney AJ, Verma DP. 1992. A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. Proceedings of the national academy of sciences USA 89: 9354-8.

Hunter IR, Walden MK, Scherer JR, Lundin RE. 1969. Preparation and properties of 1,4,5,6-tetrahydro-2-acetopyridine, a cracker-order constituent of bread aroma. Cereal Chemistry 46: 189-95.

Huyghues-Despointes A, Yaylayan VA. 1996. Retro-aldol and redox reactions of amadori compounds: mechanistic studies with variously labeled D-[13C]glucose. Journal of Agricultural and Food Chemistry 44: 672-81.

Inana G, Totsuka S, Redmond M, Dougherty T, Nagle J, Shiono T, Ohura T, Koninami E, Katunuma N. 1986. Molecular cloning oh human ornithine aminotransferase mRNA. Proceedings of the National Academy of Sciences of the United States of America 83: 1203-7.

Kalapos MP. 1999. Methylglyoxal in living organisms chemistry, biochemistry, toxicology and biological implications. Toxicology letters 110: 145-75.

Kim IJ, Lee BH, Jo J, Chung W. 2000. Isolation of a cDNA encoding a chloroplast triosephosphate isomerase from strawberry. Journal of Plant Biotehnology 2: 115-21.

Kim JS, Lee MW. 1994. Assay of ornithine aminotransferase with ninhydrin. Analytical Biochemistry 223: 205-7.

Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K, Shinozaki K. 1996. A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis. The Plant Cell 8: 1323-35.

Lebherz G, Rutter WJ. 1969. Distribution of fructose diphosphate aldolase variants in biological systems. Biochemistry. 8: 109-21.

Lin ECC, Koch JP, Chused TM, Jorgenesen SE. 1962. Utilization of L-α-glycerolphosphate by Escherichia coli without hydrolysis. Biochemistry 48: 2145-50.

Lo TW, Westwood ME, McLellan AC, Selwood T, Thornalley PJ. 1994. Binding and modification of proteins by methylglyoxal under physiological conditions. A kinetic and mechanistic study with N alpha- acetylarginine, N alpha-acetylcysteine, and N alpha-acetyllysine, and bovine serum albumin. Journal Biological Chemistry 269: 32299-305.

Mahatheeranont S, Keawsa-ard S, Dumri K. 2001. Quantification of the Rice Aroma Compound, 2-Acetyl-1-pyrroline, in Uncooked Khao Dawk Mali 105 Brown Rice. Journal of Agricultural and Food Chemistry 49: 773-9.

McLellan AC, Thornalley PJ, Benn J, Sonksen PH. 1994. Glyoxalase system in clinical diabetes mellitus and correlation with diabetic complications. Clinical Science 87: 21-9.

Minhas D, Grover A. 1999. Transcript levels of genes encoding various glycolytic and fermentation enzymes change in response to abiotic stresses. Plant Science 146: 41-51.

Misra N, Dwive UN. 1995. Carbohydrate metabolism during seed germination and seeding growth in green gram under saline stress. Plant Physiology and Biochemistry 33: 33-8.

Mottram DS. 1994. Flavor compounds formed during the Maillard reaction. American Chemical Society Washington DC 104-23.

Mounaji K, Erraiss N-E, Iddar A, Wengnez M, Serrano A, Soukri A. 2002. Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene. Comparative Biochemistry and Physiology Part B 131: 411-21.

Nakashima K, Satoh R, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K. 1998. A gene encoding proline dehydrogenase is not only induced by proline and hypoosmolarity, but is also developmentally regulated in the reproductive organs of Arabidopsis. Plant Physiology 118: 1233-41.

Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Kazuo S. 2003. Toxicity of free proline revealed in an Arabidopsis T-DNA-Tagged Mutant Deficient in proline Dehydrogenase. Plant Cell Physiol. 44: 541-8.

Nickbarg EB, Knowles JR. 1988. Triosephosphate isomerase: energetics of the reation catalyzed by the yeast enzyme expressed in Escherichia coli. Biochemistry 27: 5939-47.

Nursten HE. 1981. Recent developments in studies of the maillard reaction. Food Chemistry. 6: 263-77.

Ooi LSM, Sun SSM, Ooi VEC. 2004. Purification and characterization of a new antiviral protein from the leaves of Pandanus amaryllifolius (Pandanaceae). IGBCB 36: 1440-6.

Pelzer-Reith B, Penger A, Schnarrenberger C. 1993. Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach. Plant Molecular Biology 21: 331-40.

Pawlizki K, Latzko E. 1974. Partial separation and interconversion of NADH- and NADPH-linked activities of purified glyceraldehyde 3-phosphate dehydrogenase from spinach chloroplasts. FEBS Letter 42: 285-8.

Phillips S, Thornalley P. 1993. The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. European Journal of Biochemistry 212: 101-5.

Picklo MJ, Montine TJ, Amarnath V, Neely MD. 2002. Carbonyl toxicology and alzheimer’s disease. Toxicology and Applied Pharmacology 184: 187-97.

Pompliano DL, Peyman A, Knowles JR. 1990. Stabilization of reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase. Biochemistry 29: 3186-94.

Rajendarkumar CS, Reddy BV and Reddy AR. 1994. Proline-protein interactions: protection of structural and functional integrity of M4 lactate dehydrogenase. Biochem Biophys Res Commun 201: 957-63.

Romanczyk LJ, McClelland CA, Post LS, Aitken WM. 1995. Formation of 2-acetyl-1-pyrroline by several Bacillus cereus strains isolated from Cocoa fermentation boxes. Journal of Agricultural and Food Chemistry 43: 469-75.

Roosens NHCJ, Thu TT, Iskandar HM, Jacobs M. 1998. Isolation of the ornithine-δ-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana. Plant Physiology 117: 263-71.

Salekdeh GH, Siopongco J, Ghareyazie B, Bennet J. 2002. Proteomic analysis of rice leaves during drought stress and recovery. Proteomics 2: 1131-45.

Salim AA, Garson MJ, Craik DJ. 2004. New alkaloids from Pandanus amaryllifolius. Journal of Natural Products 67: 54-7.

Savoure A, Jaoua S, Hua XJ, Ardiles W, Van MM, Verbruggen N. 1995. Isolation, characterization, and chromosomal location of a gene encoding the Delta superior △1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana. FEBS Letters 372:13-9.

Scarpulla RC, Soffer RL. 1978. Membrane-bound proline dehydrogenase from Escherichia coli. Journal of Biological Chemistry 253: 5997-6001.

Schieberle P. 1989. Formation of 2-acetyl-1-pyrroline and other important flavor compounds in wheat bread crust. Thermal Generation of Aroma 268-275.

Schieberle P. 1990. The role of free amino acids present in yeast as precursors of the odorants 2-acetyl-1-pyrroline and 2-acetyltetrahydropyridine in wheat bread crust. Zeitschrift fur Lebensmittel-Untersuchung und-Forschung 191: 206-9.

Schieberle P. 1995. Quantitation of important roasted-smelling odorants in popcorn by stable isotope dilution assays and model studies on flavor formation during popping. Journal of Agricultural and Food Chemistry 43: 2442-8.

Schleucher J, Vanderveer PJ, Sharkeyn TD. 1998. Export of Carbon from Chloroplasts at Night. Plant Physiology 118: 1439-45.

Schultz CJ, Coruzzi G. 1995. The aspartate aminotransferase gene family of Arabidopsis thaliana encodes isoenzymes localized to three distinct subcellular compartments. The Plant Journal 7: 61-75.

Schwab W, Aharoni A, Raab T, Perez AG, Sanz C. 2001. Cytosolic aldolase is a ripening related enzyme in strawberry fruits (Fragaria × ananassa). Phytochemistry 56: 407-15.

Seitz LM, Wright RL, Waniska RD, Rooney LW. 1993. Contribution of 2-acetyl-1-pyrroline to odors from wetted ground pearl millet. Journal of Agricultural and Food Chemistry 41: 955-8.

Smirnoff N, Cumbes QJ. 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28: 1057-60.

Strecker HJ. 1971. The preparation of animal proline oxidase (rat liver), and its use for the preparation of △1-pyrroline-5-carboxylate. Methods Enzymology 17B: 251-4.

Tang GL, Wang YF, Bao JS, Chen HB. 1999. Overexpression in Escherichia coli and characterization of chloroplast triosephosphate isomerase from spinach. Protein Expression and Purification 16: 432-9.

Tang GL, Wang YF, Bao JS, Chen HB. 2001. Two-cistron system overexpression of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunit B and B-derivatives from Spinach in Escherichia coli. Protein Expression and Purification 22: 31-7.

Taylor R, Agius L. 1988. The biochemistry of diabetes. Biochemistry Journal 254: 751-5.

Thornalley PJ. 1993. The glyoxalase system in health and disease. Molecular Aspects of Medicine 14: 287-371.

Thornalley PJ. 1995. Advances in glyoxalase research. Glyoxalase expres-sion in malignancy, anti-proliferative effects of methylglyoxal, glyoxalase I inhibitor diesters and S-D-lactoylglutathione, and methyl-glyoxal modified protein binding and endocytosis by the advanced glycation endproduct receptor. Crit. Rev. Oncol. Hematol. 20: 99-128.

Thornalley PJ. 1996. Pharmacology of methylglyoxal: formation, modification of proteins and nucleic acids, and enzymatic detoxification-A role in pathogenesis and antiproliferative chemotherapy. General Pharmacology 27: 565-73.

Thornalley PJ. 1998. Glutathione-dependent detoxification of α-oxoaldehydes by the glyoxalase system: involvement in disease mechanisms and antiproliterative activity of glyoxalase Ⅰ inhibitors. Chemico-Biological Interactions 111-112: 137-151.

Tressl R, Helak B, Martin N. 1985. In: Topics in Flavor Research, Berger, RG, Nitz S, Schreier P. Eds. Verlag Hangenham: Freising-Marzling. p 139-60.

Umeda M, Uchimiya H. 1994. Differential transcript levels of gene associated with glycolysis and alcohol fermentation in rice plants (Oryza sativa L.) under submergence stress. Plant Physiology 106: 1015-22.

Venekamp JH. 1989. Regulation of cytosol acidity in plants under conditions of drought. Plant Physiology 76: 112-7.

Volpe P, Sawamura R, Strecker HJ. 1969. Control of ornithine -transaminase in rat liver and kidney. Journal of Biological Chemistry 244: 719-26.

Wong KC, Chong FN, Chee SG. 1998. Volatile constituent of taro (Colocasia esculenta (L.) Schott). The Journal of Essential Oil Research 10: 93-5.

Wongpornchai S, Sriseadka T, Choonvissase S. 2003. Identification and quantitation of the rice aroma compound, 2-acetyl-1-pyrroline, in Bread Flowers (Vallaris glabra Ktze). Journal of Agricultural and Food Chemistry 51: 457-62.

Yadav SK, Singla-Pareek SL, Ray M, Reddy MK, Sopory SK. 2005. Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochemical and Biophysical Research Communications 337: 61-7.

Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi TKS, Yamaguchi K, Wada Y, Harada Y, Shinozaki K. 1995. Correlation between the induction of a gene for delta 1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant Journal 7: 751-60.

Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-shinozaki K, Shinozaki K. 1997. Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiology 38: 1095-102.

Yoshihashi T, Huong NTT, Inatomi H. 2002. Precursors of 2-acetyl -1-pyrroline, a potent flavor compound of an aromatic rice variety. Journal of Agricultural and Food Chemistry 50: 2001-4.