Identification of rice ethylene-response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation

Molecular Plant

Volumn 6, Issue 6, 2013, Pages 1830-1848

  Ma, Biao ^a   He, Si Jie ^a   Duan, Kai Xuan ^a   Yin, Cui Cui ^a   Chen, Hui ^a   Yang, Chao ^a   Xiong, Qing ^a   Song, Qing Xin ^a   Lu, Xiang ^a   Chen, Hao Wei ^a   Zhang, Wan Ke ^a   Lu, Tie Gang ^b   Chen, Shou Yi ^a   Zhang, Jin Song ^a  

^a INSTITUTE OF GENETICS AND DEVELOPMENTAL BIOLOGY   (China)

^b INSTITUTE OF PLANT PROTECTION   (China)

Author keywords

Ethylene response mutant; MHZ7; Rice; Senescence; Yield traits

Indexed keywords

BETA GLUCURONIDASE; ETHYLENE; ETHYLENE DERIVATIVE; VEGETABLE PROTEIN;

ARTICLE; ETHYLENE-RESPONSE MUTANT; ETIOLATION; GENE EXPRESSION REGULATION; GENETICS; GROWTH, DEVELOPMENT AND AGING; METABOLISM; MHZ7; MUTATION; PLANT LEAF; PLANT ROOT; PROMOTER REGION; RICE; SENESCENCE.; YIELD TRAITS;

ETHYLENE-RESPONSE MUTANT; MHZ7; RICE; SENESCENCE.; YIELD TRAITS;

ETHYLENES; ETIOLATION; GENE EXPRESSION REGULATION, PLANT; GLUCURONIDASE; MUTATION; ORYZA SATIVA; PLANT LEAVES; PLANT PROTEINS; PLANT ROOTS; PROMOTER REGIONS, GENETIC;

EID: 84888874497     PISSN: 16742052     EISSN: 17529867     Source Type: Journal    
DOI: 10.1093/mp/sst087     Document Type: Article

References (86)

1. Alonso, J.M., Hirayama, T., Roman, G., Nourizadeh, S., and Ecker, J.R. (1999). EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 284, 2148-2152.
2. An, F., Zhao, Q., Ji, Y., Li, W., Jiang, Z., Yu, X., Zhang, C., Han, Y., He, W., Liu, Y., et al. (2010). Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. Plant Cell. 22, 2384-2401.
3. Binder, B.M., O'Malley, R.C., Wang, W., Moore, J.M., Parks, B.M., Spalding, E.P., and Bleecker, A.B. (2004). Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis. Plant Physiol. 136, 2913-2920.
4. Binder, B.M., Walker, J.M., Gagne, J.M., Emborg, T.J., Hemmann, G., Bleecker, A.B., and Vierstra, R.D. (2007). The Arabidopsis EIN3 binding F-Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling. Plant Cell. 19, 509-523.
5. Bleecker, A.B., and Kende, H. (2000). Ethylene: A gaseous signal molecule in plants. Annu. Rev. Cell Dev. Biol. 16, 1-18.
6. Cao, W.H., Dong, Y., Zhang, J.S., and Chen, S.Y. (2003). Characterization of an ethylene receptor homolog gene from rice. Sci. China C Ser. Life Sci. 46, 370-378.
7. Cao, W.H., Liu, J., He, X.J., Mu, R.L., Zhou, H.L., Chen, S.Y., and Zhang, J.S. (2007). Modulation of ethylene responses affects plant salt-stress responses. Plant Physiol. 143, 707-719.
8. Cao, W.H., Liu, J., Zhou, Q.Y., Cao, Y.R., Zheng, S.F., Du, B.X., Zhang, J.S., and Chen, S.Y. (2006). Expression of tobacco ethylene receptor NTHK1 alters plant responses to salt stress. Plant, Cell Environ. 29, 1210-1219.
9. Cervantes, E., Javier Martín, J., Ardanuy, R., de Diego, J.G., and Tocino, Á. (2010). Modeling the Arabidopsis seed shape by a oleoptil: Efficacy of the adjustment with a scale change with factor equal to the Golden Ratio and analysis of seed shape in ethylene mutants. J. Plant Physiol. 167, 408-410.
10. Cervantes, E., Martín, J.J., Chan, P.K., Gresshoff, P.M., and Tocino, Á. (2012). Seed shape in model legumes: Approximation by a oleoptil reveals differences in ethylene insensitive mutants of Lotus oleoptil and Medicago truncatula. J. Plant Physiol. 169, 1359-1365.
11. Chang, C., Kwok, S.F., Bleecker, A.B., and Meyerowitz, E.M. (1993). Arabidopsis ethylene-response gene ETR1: Similarity of product to two-component regulators. Science. 262, 539-544.
12. Chao, Q., Rothenberg, M., Solano, R., Roman, G., Terzaghi, W., and Ecker, J.R. (1997). Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENEINSENSITIVE3 and related proteins. Cell. 89, 1133-1144.
13. Chen, T., Liu, J., Lei, G., Liu, Y.F., Li, Z.G., Tao, J.J., Hao, Y.J., Cao, Y.R., Lin, Q., Zhang, W.K., et al. (2009). Effects of tobacco ethylene receptor mutations on receptor kinase activity, plant growth and stress responses. Plant Cell Physiol. 50, 1636-1650.
14. Chen, Y.F., Shakeel, S.N., Bowers, J., Zhao, X.C., Etheridge, N., and Schaller, G.E. (2007). Ligand-induced degradation of the ethylene receptor ETR2 through a proteasome-dependent pathway in Arabidopsis. J. Biol. Chem. 282, 24752-24758.
15. Fukao, T., and Bailey-Serres, J. (2008). Ethylene-A key regulator of submergence responses in rice. Plant Sci. 175, 43-51.
16. Fukao, T., Xu, K., Ronald, P.C., and Bailey-Serres, J. (2006). A variable cluster of ethylene response factor-like genes regulates metabolic and developmental acclimation responses to submergence in rice. Plant Cell. 18, 2021-2034.
17. Gagne, J.M., Smalle, J., Gingerich, D.J., Walker, J.M., Yoo, S.D., Yanagisawa, S., and Vierstra, R.D. (2004). Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation. Proc. Natl Acad. Sci. U S A. 101, 6803-6808.
18. Gamble, R.L., Coonfield, M.L., and Schaller, G.E. (1998). Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis. Proc. Natl Acad. Sci. U S A. 95, 7825-7829.
19. Guo, H., and Ecker, J.R. (2003). Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor. Cell. 115, 667-77.
20. Hall, B.P., Shakeel, S.N., Amir, M., Ul Haq, N., Qu, X., and Schaller, G.E. (2012). Histidine kinase activity of the ethylene receptor ETR1 facilitates the ethylene response in Arabidopsis. Plant Physiol. 159, 682-695.
21. Hattori, Y., Nagai, K., Furukawa, S., Song, X.J., and Kawano, R. (2009). The ethylene response factors Snorkel1 and Snorkel2 allow rice to adapt to deep water. Nature. 460, 1026-1030.
22. He, X.J., Mu, R.L., Cao, W.H., Zhang, Z.G., Zhang, J.S., and Chen, S.Y. (2005). AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J. 44, 903-916.
23. Hua, J., and Meyerowitz, E.M. (1998). Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell. 94, 261-271.
24. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995). Ethylene insensitivity conferred by Arabidopsis ERS gene. Science. 269, 1712-1714.
25. Hua, J., Sakai, H., Nourizadeh, S., Chen, Q.G., Bleecker, A.B., Ecker, J.R., and Meyerowitz, E.M. (1998). EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. Plant Cell. 10, 1321-1332.
26. Itoh, J., Nonomura, K., and Ikeda, K. (2005). Rice plant development: From zygote to spikelet. Plant Cell Physiol. 46, 23-47.
27. Jackson, M.B. (2008). Ethylene-promoted elongation: An adaptation to submergence stress. Ann. Bot. 101, 229-248.
28. Ju, C., Yoon, G.M., Shemansky, J.M., Lin, D.Y., Ying, Z.I., Chang, J., Garrett, W.M., Kessenbrock, M., Groth, G., Tucker, M.L., et al. (2012). CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc. Natl Acad. Sci. U S A. 109, 19486-19491.
29. Jun, S.H., Han, M.J., Lee, S., Seo, Y.S., Kim, W.T., and An, G. (2004). OsEIN2 is a positive component in ethylene signaling in rice. Plant Cell Physiol. 45, 281-289.
30. Kamiyoshihara, Y., Tieman, D.M., Huber, D.J., and Klee, H.J. (2012). Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit. Plant Physiol. 60, 488-497.
31. Kevany, B.M., Tieman, D.M., Taylor, M.G., Cin, V.D., and Klee, H.J. (2007). Ethylene receptor degradation controls the timing of ripening in tomato fruit. Plant J. 51, 458-467.
32. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993). CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell. 72, 427-41.
33. Kim, H., Helmbrecht, E.E., Stalans, M.B., Schmitt, C., Patel, N., Wen, C.K., Wang, W., and Binder, B.M. (2011). Ethylene receptor ETHYLENE RECEPTOR1 domain requirements for ethylene responses in Arabidopsis seedlings. Plant Physiol. 156, 417-429.
34. Kim, J., Wilson, R.L., Case, J.B., and Binder, B.M. (2012). Ethylene growth response kinetics in eudicots and monocots reveals a role for gibberellin in growth inhibition and recovery. Plant Physiol. 160, 1567-1580.
35. Kim, J.H., Woo, H.R., Kim, J., Lim, P.O., Lee, I.C., Choi, S.H., Hwang, D., and Nam, H.G. (2009). Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science. 323, 1053-1057.
36. Ku, H.S., Suge, H., Rappaport, L., and Pratt, H.K. (1970). Stimulation of rice coleoptile growth by ethylene. Planta. 90, 333-339.
37. Lee, R.H., Wang, C.H., Huang, L.T., and Chen, S.C.G. (2000). Leaf senescence in rice plants: Cloning and characterization of senescence up-regulated genes. J. Exp. Bot. 52, 1117-11212.
38. Lei, G., Shen, M., Li, Z.G., Zhang, B., Duan, K.X., Wang, N., Cao, Y.R., Zhang, W.K., Ma, B, Ling, H.Q., et al. (2011). EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis. Plant Cell Environ. 34, 1678-1692.
39. Ma, B., Chen, S.Y., and Zhang, J.S. (2010). Ethylene signaling in rice. Chin. Sci. Bull. 55, 2204-2210.
40. Mao, C., Wang, S., Jia, Q., and Wu, P. (2006). OsEIL1, a rice homolog of the Arabidopsis EIN3 regulates the ethylene response as a positive component. Plant Mol. Biol. 61, 141-152.
41. Michelmore, R.W., Paran, I., and Kesseli, R.V. (1991). Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl Acad. Sci. U S A. 88, 9828-9832.
42. Moussatche, P., and Klee, H.J. (2004). Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family. J. Biol. Chem. 279, 48734-48741.
43. O'Malley, R.C., Rodriguez, F.I., Esch, J.J., Binder, B.M., O'Donnell, P., Klee, H.J., and Bleecker, A.B. (2005). Ethylene-binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato. Plant J. 41, 651-659.
44. Peng, H., Huang, H., Yang, Y., Zhai, Y., Wu, J., Huang, D., and Lu, T. (2005). Functional analysis of GUS expression patterns and T-DNA integration characteristics in rice enhancer trap lines. Plant Sci. 168, 1571-1579.
45. Penmetsa, R.V., Uribe, P., Anderson, J., Lichtenzveig, J., Gish, J.C., Nam, Y.W., Engstrom, E., Xu, K., Sckisel, G., and Pereira, M. (2008). The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations. Plant J. 55, 580-595.
46. Potuschak, T., Lechner, E., Parmentier, Y., Yanagisawa, S., Grava, S., Koncz, C., and Genschik, P. (2003). EIN3-dependent regulation of plant ethylene hormone signaling by two Arabidopsis F box proteins: EBF1 and EBF2. Cell. 115, 679-689.
47. Qiao, H., Chang, K.N., Yazaki, J., and Ecker, J.R. (2009). Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. 23, 512-521.
48. Qiao, H., Shen, Z., Huang, S.S., Schmitz, R.J., Urich, M.A., Briggs, S.P., and Ecker, J.R. (2012). Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science. 338, 390-393.
49. Qiu, L., Xie, F., Yu, J., and Wen, C.K. (2012). Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1. Plant Physiol. 159, 1263-1276.
50. Qu, X., and Schaller, G.E. (2004). Requirement of the histidine kinase domain for signal transduction by the ethylene receptor ETR1. Plant Physiol. 136, 2961-2970.
51. Resnick, J.S., Rivarola, M., and Chang, C. (2008). Involvement of RTE1 in conformational changes promoting ETR1 ethylene receptor signaling in Arabidopsis. Plant J. 56, 423-431.
52. Resnick, J.S., Wen, C.K., Shockey, J.A., and Chang, C. (2006). REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis. Proc. Natl Acad. Sci. U S A. 103, 7917-7922.
53. Robert, C., Noriega, A., Tocino, A., and Cervantes, E. (2008). Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway. J. Plant Physiol. 165, 911-919.
54. Rzewuski, G., and Sauter, M. (2008). Ethylene biosynthesis and signaling in rice. Plant Sci. 175, 32-42
55. Sakai, H., Hua, J., Chen, Q.G., Chang, C., Medrano, L.J., Bleecker, A.B., and Meyerowitz, E.M. (1998). ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis. Proc. Natl Acad. Sci. U S A. 95, 5812-5817.
56. Schaller, G.E., and Bleecker, A.B. (1995). Ethylene-binding sites generated in yeast expressing the Arabidopsis ETR1 gene. Science. 270, 1809-1811.
57. Serek, M., Tamari, G., Sisler, E.C., and Borochov, A. (1995). Inhibition of ethylene-induced cellular senescence symptoms by 1-methylcyclopropene, a new inhibitor of ethylene action. Physiol. Plant. 94, 229-232.
58. Shi, Y., Tian, S., Hou, L., Huang, X., Zhang, X., Guo, H., and Yang, S. (2012). Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell. 24, 2578-2595.
59. Shibuya, K., Barry, K.G., Ciardi, J.A., Loucas, H.M., Underwood, B.A., Nourizadeh, S., Ecker, J.R., Klee, H.J., and Clark, D.G. (2004). The central role of PhEIN2 in ethylene responses throughout plant development in petunia. Plant Physiol. 136, 2900-2912.
60. Sisler, E.C., and Serek, M. (2003). Compounds interacting with the ethylene receptor in plants. Plant Biol. 5, 473-80.
61. Solano, R., Stepanova, A., Chao, Q., and Ecker, J.R. (1998). Nuclear events in ethylene signaling: A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENERESPONSE-FACTOR1. Genes Dev. 12, 3703-3714.
62. Steffens, B., and Sauter, M. (2009). Epidermal cell death in rice is confined to cells with a distinct molecular identity and is mediated by ethylene and H2O2 through an autoamplified signal pathway. Plant Cell. 21, 184-196.
63. Steffens, B., Kovalev, A., Gorb, S.N., and Sauter, M. (2012). Emerging roots alter epidermal cell fate through mechanical and reactive oxygen species signaling. Plant Cell. 24, 3296-3306.
64. Tieman, D.M., Taylor, M.G., Ciardi, J.A., and Klee, H.J. (2000). The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proc. Natl Acad. Sci. U S A. 97, 5663-5668.
65. Walton, L.J., Kurepin, L.V., Yeung, E.C., Shah, S., Emery, R.J., Reid, D.M., and Pharis, R.P. (2012). Ethylene involvement in silique and seed development of canola, Brassica napus L. Plant Physiol. Biochem. 58, 142-150.
66. Wan, S., Wu, J., Zhang, Z., Sun, X., Lv, Y., Gao, C., Ning, Y., Ma, J., Guo, Y., Zhang, Q., et al. (2009). Activation tagging, an efficient tool for functional analysis of the rice genome. Plant Mol. Biol. 69, 69-80.
67. Wang, W., Esch, J.J., Shiu, S.H., Agula, H., Binder, B.M., Chang, C., Patterson, S.E., and Bleecker, A.B. (2006). Identification of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis. Plant Cell. 18, 3429-3442.
68. Wang, W., Hall, A.E., O'Malley, R., and Bleecker, A.B. (2003). Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission. Proc. Natl Acad. Sci. U S A. 100, 352-357.
69. Watanabe, H., Saigusa, M., Hase, S., Hayakawa, T., and Satoh, S. (2004). Cloning of a cDNA encoding an ETR2-like protein (Os-ERL1) from deep water rice (Oryza sativa L.) and increase in its mRNA level by submergence, ethylene, and gibberellin treatments. J. Exp. Bot. 55, 1145-1148.
70. Wen, X., Zhang, C., Ji, Y., Zhao, Q., He, W., An, F., Jiang, L., and Guo, H. (2012). Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus. Cell Res. 22, 1613-1616.
71. Wuriyanghan, H., Zhang, B., Cao, W.H., Ma, B., Lei, G., Liu, Y.F., Wei, W., Wu, H.J., Chen, L.J., Chen, H.W., et al. (2009). The ethylene receptor ETR2 delays floral transition and affects starch accumulations in rice. Plant Cell. 21, 1473-1494.
72. Xie, C., Zhang, J.S., Zhou, H.L., Li, J., Zhang, Z.G., Wang, D.W., and Chen, S.Y. (2003). Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. Plant J. 33, 385-393.
73. Xie, F., Liu, Q., and Wen, C.K. (2006). Receptor signal output mediated by the ETR1 N terminus is primarily subfamily I receptor dependent. Plant Physiol. 142, 492-508.
74. Xie, F., Qiu, L., and Wen, C.K. (2012). Possible modulation of Arabidopsis ETR1 N-terminal signaling by CTR1. Plant Signal. Behav. 7, 1243-1245.
75. Xu, K., Xu, X., Fukao, T., Canlas, P., Maghirang-Rodriguez, R., Heuer, S., Ismail, A.M., Bailey-Serres, J., Ronald, P.C., and Mackill, D.J. (2006). Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature. 442, 705-708.
76. Yang, Y., Peng, H., Huang, H., Wu, J., Jia, S., Huang, D., and Lu, T. (2004). Large-scale production of enhancer trapping lines for rice functional genomics. Plant Sci. 167, 281-288.
77. Yau, C.P., Wang, L., Yu, M., Zee, S.Y., and Yip, W.K. (2004). Differential expression of three genes encoding an ethylene receptor in rice during development, and in response to indole-3-acetic acid and silver ions. J. Exp. Bot. 55, 547-556.
78. Zhang, B., Chen, H.W., Mu, R.L., Zhang, W.K., Zhao, M.Y., Wei, W., Wang, F., Yu, H., Lei, G., Zou, H.F., et al. (2011a). NIMArelated kinase NEK6 affects plant growth and stress response in Arabidopsis. Plant J. 68, 830-843.
79. Zhang, L., Li, Z., Quan, R., Li, G., Wang, R., and Huang, R. (2011b). An AP2 domain-containing gene, ESE1, targeted by the ethylene signaling component EIN3 is important for the salt response in Arabidopsis. Plant Physiol. 157, 854-865.
80. Zhang, W., Zhou, X., and Wen, C.K. (2012). Modulation of ethylene responses by OsRTH1 overexpression reveals the biological significance of ethylene in rice seedling growth and development. J. Exp. Bot. 63, 4151-4164.
81. Zhang, Z.G., Zhou, H.L., Chen, T., Gong, Y., Cao, W.H., Wang, Y.J., Zhang, J.S., and Chen, S.Y. (2004). Evidence for serine/threonine and histidine kinase activity in the tobacco ethylene receptor protein NTHK2. Plant Physiol. 136, 2971-2981.
82. Zhong, S., Shi, H., Xue, C., Wang, L., Xi, Y., Li, J., Quail, P.H., Deng, X.W., and Guo, H. (2012). A molecular framework of lightcontrolled phytohormone action in Arabidopsis. Curr. Biol. 22, 1530-1535.
83. Zhong, S., Zhao, M., Shi, T., Shi, H., An, F., Zhao, Q., and Guo, H. (2009). EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings. Proc. Natl Acad. Sci. U S A. 106, 21431-21436.
84. Zhou, H.L., Cao, W.H., Cao, Y.R., Liu, J., Hao, Y.J., Zhang, J.S., and Chen, S.Y. (2006). Roles of ethylene receptor NTHK1 domains in plant growth, stress response and protein phosphorylation. FEBS Lett. 580, 1239-1250.
85. Zhou, X., Liu, Q., Xie, F., and Wen, C.K. (2007). RTE1 is a Golgiassociated and ETR1-dependent negative regulator of ethylene responses. Plant Physiol. 145, 75-86.
86. Zhu, Z., An, F., Feng, Y., Li, P., Xue, L., A M, Jiang, Z., Kim, J.M., To, T.K., Li, W., et al. (2011). Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. Proc. Natl Acad. Sci. U S A. 108, 12539-12544.