植物非專一性脂質傳送蛋白 (nsLTP)是一類分子量小且穩定的蛋白，這類蛋白依照分子量大小被分為兩大類：即nsLTP1 (9 kDa)以及nsLTP2 (7 kDa)。到目前為止，nsLTPs被發現具有多種生物功能，包括：結合與傳送脂質、抑制α-澱粉酶、抑制蛋白酶以及抗菌的能力。近期在阿拉伯芥中發現花粉專一性表現的酸性nsLTP (AAtLTP)，而它的功能、結構與特性在目前還是未知。因此我們將在此篇中研究AAtLTP的功能與基礎結構並與另一個鹼性nsLTP (LTP2)進行比較。在二級結構的研究上，AAtLTP及LTP2是非常不同的，而它們在高溫與極酸及極鹼環境下都表現高度穩定性。在本篇實驗中顯示AAtLTP及LTP2都不具有抑制α-澱粉酶及抑制蛋白酶的能力。不過LTP2可以有效的抑制鮑氏不動桿菌(Acinetobacter baumanni)與白色念珠菌(Candida albicans)的生長，而AAtLTP則不具抗菌能力。另外LTP2對於結合脂肪酸及固醇的能力很好，而且也可以有效的將磷脂質在兩個膜之間做傳送。雖然AAtLTP沒有以上的結合與傳送能力，但AAtLTP對於香豆酸(ρ-coumaric acid)有良好的結合能力，且結合活性高於LTP2。ρ-coumaric acid是合成花粉外壁的重要成分，這讓我們聯想AAtLTP對於花粉的形成與發育是具有影響的。

Palnt nonspecific lipid transfer proteins (nsLTPs) are small and stable proteins, which are classified into two subfamilies by molecular weight: nsLTP1 (9 kDa) and nsLTP2 (7 kDa). Currently, nsLTPs are known to have the ability to bind and transfer lipid between membranes, inhibit α-amylase activity, inhibit protease activity and display antimicrobial activity. Recently, an anther-specific acidic nsLTP (AAtLTP) has been found in Arabidopsis thaliana, while its biophysical and biochemical properties is still unknown. Therefore, in this study, we focused on structural and functional properties of AAtLTP and compared these results with the basic nsLTP (LTP2). By circular dichroism, the secondary structure of AAtLTP and LTP2 are very different, but both exhibited strong stabilities on high temperature (up to 80 ˚C) and broad pH (pH 3 to pH 13) range. On the other hand, some tests were performed to discover the biological function of AAtLTP. We found that AAtLTP and LTP2 both could not inhibit α-amylase and trypsin enzyme activities. In anti-microbial experiments, LTP2 exhibited good anti-bacterial and anti-fungal activities, against Acinetobacter baumanni and Candida albicans, but AAtLTP doesn’t have the anti-microbial ability. Moreover, the fluorescence binding assay was also used to further investigate the lipid binding abilities of nsLTPs. The results showed that LTP2 could bind to fatty acid, sterol and ρ-coumaric acid while AAtLTP could only bind to ρ-coumaric acid but with higher binding affinity than LTP2. The AAtLTP can bind ρ-coumaric acid, the lipid molecule on pollen surface, implying that this acidic nsLTP is potentially involved in the anther development. In this work, we investigated the biophysical properties and biological characters of AAtLTP, and the lipid binding selectivity and compared these results with basic nsLTP2 to further insight into the relationship between function and structure of plant LTP.

1. Liu, Y. J., Samuel, D., Lin, C. H., and Lyu, P. C. (2002) Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa), Biochemical and biophysical research communications 294, 535-540.
2. Boutrot, F., Chantret, N., and Gautier, M. F. (2008) Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein (nsLtp) gene families and identification of wheat nsLtp genes by EST data mining, BMC genomics 9, 86.
3. Douliez, J. P., Jegou, S., Pato, C., Larre, C., Molle, D., and Marion, D. (2001) Identification of a new form of lipid transfer protein (LTP1) in wheat seeds, Journal of agricultural and food chemistry 49, 1805-1808.
4. Lin, K. F., Liu, Y. N., Hsu, S. T., Samuel, D., Cheng, C. S., Bonvin, A. M., and Lyu, P. C. (2005) Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean, Biochemistry 44, 5703-5712.
5. Shin, D. H., Lee, J. Y., Hwang, K. Y., Kim, K. K., and Suh, S. W. (1995) High-resolution crystal structure of the non-specific lipid-transfer protein from maize seedlings, Structure 3, 189-199.
6. Horvath, B. M., Bachem, C. W., Trindade, L. M., Oortwijn, M. E., and Visser, R. G. (2002) Expression analysis of a family of nsLTP genes tissue specifically expressed throughout the plant and during potato tuber life cycle, Plant physiology 129, 1494-1506.
7. Douliez, J. P., Michon, T., Elmorjani, K., and Marion, D. (2000) Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels, J Cereal Sci 32, 1-20.
8. Kader, J. C. (1996) Lipid-Transfer Proteins in Plants, Annual review of plant physiology and plant molecular biology 47, 627-654.
9. Douliez, J. P., Michon, T., and Marion, D. (2000) Steady-state tyrosine fluorescence to study the lipid-binding properties of a wheat non-specific lipid-transfer protein (nsLTP1), Biochimica et biophysica acta 1467, 65-72.
10. Park, S. Y., Jauh, G. Y., Mollet, J. C., Eckard, K. J., Nothnagel, E. A., Walling, L. L., and Lord, E. M. (2000) A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix, The Plant cell 12, 151-164.
11. Nieuwland, J., Feron, R., Huisman, B. A., Fasolino, A., Hilbers, C. W., Derksen, J., and Mariani, C. (2005) Lipid transfer proteins enhance cell wall extension in tobacco, The Plant cell 17, 2009-2019.
12. Edqvist, J., and Farbos, I. (2002) Characterization of germination-specific lipid transfer proteins from Euphorbia lagascae, Planta 215, 41-50.
13. Gonorazky, A. G., Regente, M. C., and de la Canal, L. (2005) Stress induction and antimicrobial properties of a lipid transfer protein in germinating sunflower seeds, Journal of plant physiology 162, 618-624.
14. Hincha, D. K., Neukamm, B., Sror, H. A., Sieg, F., Weckwarth, W., Ruckels, M., Lullien-Pellerin, V., Schroder, W., and Schmitt, J. M. (2001) Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family, Plant physiology 125, 835-846.
15. Cameron, K. D., Teece, M. A., and Smart, L. B. (2006) Increased accumulation of cuticular wax and expression of lipid transfer protein in response to periodic drying events in leaves of tree tobacco, Plant physiology 140, 176-183.
16. Jung, H. W., Kim, K. D., and Hwang, B. K. (2005) Identification of pathogen-responsive regions in the promoter of a pepper lipid transfer protein gene (CALTPI) and the enhanced resistance of the CALTPI transgenic Arabidopsis against pathogen and environmental stresses, Planta 221, 361-373.
17. Sohal, A. K., Pallas, J. A., and Jenkins, G. I. (1999) The promoter of a Brassica napus lipid transfer protein gene is active in a range of tissues and stimulated by light and viral infection in transgenic Arabidopsis, Plant molecular biology 41, 75-87.
18. Molina, A., Segura, A., and Garcia-Olmedo, F. (1993) Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens, FEBS letters 316, 119-122.
19. Terras, F. R., Schoofs, H. M., De Bolle, M. F., Van Leuven, F., Rees, S. B., Vanderleyden, J., Cammue, B. P., and Broekaert, W. F. (1992) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds, The Journal of biological chemistry 267, 15301-15309.
20. Diz, M. S., Carvalho, A. O., Ribeiro, S. F., Da Cunha, M., Beltramini, L., Rodrigues, R., Nascimento, V. V., Machado, O. L., and Gomes, V. M. (2011) Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties, Physiologia plantarum 142, 233-246.
21. Zottich, U., Da Cunha, M., Carvalho, A. O., Dias, G. B., Silva, N. C., Santos, I. S., do Nacimento, V. V., Miguel, E. C., Machado, O. L., and Gomes, V. M. (2011) Purification, biochemical characterization and antifungal activity of a new lipid transfer protein (LTP) from Coffea canephora seeds with alpha-amylase inhibitor properties, Biochimica et biophysica acta 1810, 375-383.
22. Scheurer, S., Lauer, I., Foetisch, K., San Miguel Moncin, M., Retzek, M., Hartz, C., Enrique, E., Lidholm, J., Cistero-Bahima, A., and Vieths, S. (2004) Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion, The Journal of allergy and clinical immunology 114, 900-907.
23. Lehmann, M. S., Pebay-Peyroula, E., Cohen-Addad, C., and Odani, S. (1989) Crystallographic data for soybean hydrophobic protein, Journal of molecular biology 210, 235-236.
24. Wang, N. J., Lee, C. C., Cheng, C. S., Lo, W. C., Yang, Y. F., Chen, M. N., and Lyu, P. C. (2012) Construction and analysis of a plant non-specific lipid transfer protein database (nsLTPDB), BMC genomics 13 Suppl 1, S9.
25. Lee, J. Y., Min, K., Cha, H., Shin, D. H., Hwang, K. Y., and Suh, S. W. (1998) Rice non-specific lipid transfer protein: the 1.6 A crystal structure in the unliganded state reveals a small hydrophobic cavity, Journal of molecular biology 276, 437-448.
26. Han, G. W., Lee, J. Y., Song, H. K., Chang, C., Min, K., Moon, J., Shin, D. H., Kopka, M. L., Sawaya, M. R., Yuan, H. S., Kim, T. D., Choe, J., Lim, D., Moon, H. J., and Suh, S. W. (2001) Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography, Journal of molecular biology 308, 263-278.
27. Samuel, D., Liu, Y. J., Cheng, C. S., and Lyu, P. C. (2002) Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa), The Journal of biological chemistry 277, 35267-35273.
28. Douliez, J. P., Pato, C., Rabesona, H., Molle, D., and Marion, D. (2001) Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2, European journal of biochemistry / FEBS 268, 1400-1403.
29. Lerche, M. H., Kragelund, B. B., Bech, L. M., and Poulsen, F. M. (1997) Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands, Structure 5, 291-306.
30. Cheng, C. S., Samuel, D., Liu, Y. J., Shyu, J. C., Lai, S. M., Lin, K. F., and Lyu, P. C. (2004) Binding mechanism of nonspecific lipid transfer proteins and their role in plant defense, Biochemistry 43, 13628-13636.
31. Cheng, C. S., Chen, M. N., Lai, Y. T., Chen, T., Lin, K. F., Liu, Y. J., and Lyu, P. C. (2008) Mutagenesis study of rice nonspecific lipid transfer protein 2 reveals residues that contribute to structure and ligand binding, Proteins 70, 695-706.
32. Yang, H., Lu, P., Wang, Y., and Ma, H. (2011) The transcriptome landscape of Arabidopsis male meiocytes from high-throughput sequencing: the complexity and evolution of the meiotic process, The Plant journal : for cell and molecular biology 65, 503-516.
33. Nakamura, S., Suzuki, T., Kawamukai, M., and Nakagawa, T. (2012) Expression analysis of Arabidopsis thaliana small secreted protein genes, Bioscience, biotechnology, and biochemistry 76, 436-446.
34. Peiffer, J. A., Kaushik, S., Sakai, H., Arteaga-Vazquez, M., Sanchez-Leon, N., Ghazal, H., Vielle-Calzada, J. P., and Meyers, B. C. (2008) A spatial dissection of the Arabidopsis floral transcriptome by MPSS, BMC plant biology 8, 43.
35. Wijeratne, A. J., Zhang, W., Sun, Y., Liu, W., Albert, R., Zheng, Z., Oppenheimer, D. G., Zhao, D., and Ma, H. (2007) Differential gene expression in Arabidopsis wild-type and mutant anthers: insights into anther cell differentiation and regulatory networks, The Plant journal : for cell and molecular biology 52, 14-29.
36. Feng, B., Lu, D., Ma, X., Peng, Y., Sun, Y., Ning, G., and Ma, H. (2012) Regulation of the Arabidopsis anther transcriptome by DYT1 for pollen development, The Plant journal : for cell and molecular biology 72, 612-624.
37. Ma, X., Feng, B., and Ma, H. (2012) AMS-dependent and independent regulation of anther transcriptome and comparison with those affected by other Arabidopsis anther genes, BMC plant biology 12, 23.
38. van Paridon, P. A., Gadella, T. W., Jr., Somerharju, P. J., and Wirtz, K. W. (1988) Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain, Biochemistry 27, 6208-6214.
39. Pei-pei, M., Li-xia, Z., Ri-wei, W., Xu-dong, Q., and Qi, Y. (2009) Fluorescent characteristic of ρ-coumaric acid, caffenic acid and chlorogenic aicd, Journal of Shandong Agricultural University (Natural Science) 40.
40. Strobl, S., Gomis-Ruth, F. X., Maskos, K., Frank, G., Huber, R., and Glockshuber, R. (1997) The alpha-amylase from the yellow meal worm: complete primary structure, crystallization and preliminary X-ray analysis, FEBS letters 409, 109-114.
41. Kazzazi, M., Bandani, A. R., Ashuri, A., and Hosseinkhani, S. (2005) A amylase activity of nymphal stages of sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), Communications in agricultural and applied biological sciences 70, 863-867.
42. Tsai, P. W., Yang, C. Y., Chang, H. T., and Lan, C. Y. (2011) Human antimicrobial peptide LL-37 inhibits adhesion of Candida albicans by interacting with yeast cell-wall carbohydrates, PloS one 6, e17755.
43. Morant, M., Jorgensen, K., Schaller, H., Pinot, F., Moller, B. L., Werck-Reichhart, D., and Bak, S. (2007) CYP703 is an ancient cytochrome P450 in land plants catalyzing in-chain hydroxylation of lauric acid to provide building blocks for sporopollenin synthesis in pollen, The Plant cell 19, 1473-1487.