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Plant function and evolutionary biology
RESEARCH ARTICLE

UV-B-induced stomatal closure occurs via ethylene-dependent NO generation in Vicia faba

Jun-Min He A C , Zhan Zhang A , Rui-Bin Wang A and Yi-Ping Chen B
+ Author Affiliations
- Author Affiliations

A School of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an 710062, People’s Republic of China.

B Institute of Earth Environment, Chinese Academy of Sciences, Xi’an 710075, People’s Republic of China.

C Corresponding author. Email: hejm@snnu.edu.cn

Functional Plant Biology 38(4) 293-302 https://doi.org/10.1071/FP10219
Submitted: 18 November 2010  Accepted: 8 February 2011   Published: 8 April 2011

Abstract

The role of ethylene and the relationship between ethylene and nitric oxide (NO) in ultraviolet B (UV-B)-induced stomatal closure were investigated in Vicia faba L. (broad bean) plants by epidermal strip bioassay, laser-scanning confocal microscopy and assay of ethylene production. In response to UV-B radiation, the rise of NO level in guard cells was after ethylene evolution peak, but preceded stomatal closure. Both UV-B-induced NO generation in guard cells and subsequent stomatal closure were substantially inhibited not only by NO scavenger and nitrate reductase (NR) inhibitors, but also by interfering with ethylene synthesis or perception. Although exogenous NO could reverse the inhibitive effect of interfering with ethylene synthesis or perception on UV-B-induced stomatal closure, the inhibitive effect of NO scavenger and NR inhibitors on UV-B-induced stomatal closure could not be rescued by exogenous ethylene. Taken together, our results clearly show that ethylene participates in the UV-B-induced stomatal closure and acts upstream of the NR source of NO generation in V. faba.

Additional keywords: ethene, nitric oxide generation, stomatal movement, UV-B radiation, Vicia faba.


References

Acharya BR, Assmann SM (2009) Hormone interactions in stomatal function. Plant Molecular Biology 69, 451–462.
Hormone interactions in stomatal function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhvVOisL8%3D&md5=20339fe82bf23c7a53e15711f917a429CAS | 19031047PubMed |

Adams DO, Yang SF (1979) Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proceedings of the National Academy of Sciences of the United States of America 76, 170–174.
Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1MXhtFyisLo%3D&md5=5d904885bb59e15a32214e6df876ea8bCAS | 16592605PubMed |

An L, Liu Y, Zhang M, Chen T, Wang X (2005) Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation. Journal of Plant Physiology 162, 317–326.
Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXktlanuro%3D&md5=6ca89bfd2b3dc25f85a0b994fd41185bCAS | 15832684PubMed |

An L, Xu X, Tang H, Zhang M, Hou Z, Liu Y, Zhao Z, Feng H, Xu S, Wang X (2006) Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase gene expression in tomato (Lycopsicon esculentum Mill.) leaves under enhanced UV-B radiation. Journal of Integrative Plant Biology 48, 1190–1196.
Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase gene expression in tomato (Lycopsicon esculentum Mill.) leaves under enhanced UV-B radiation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xht1Sqs77E&md5=2415ade7bb5f0a67222de70cf85afc27CAS |

Arnaud N, Murgia I, Boucherez J, Briat J-F, Cellier F, Gaymard F (2006) An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression. Journal of Biological Chemistry 281, 23 579–23 588.
An iron-induced nitric oxide burst precedes ubiquitin-dependent protein degradation for Arabidopsis AtFer1 ferritin gene expression.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XotVCmtLY%3D&md5=27f71a8a80c2f39e04d2381bc8812992CAS |

Benlloch-González M, Romera J, Cristescu S, Harren F, Fournier JM, Benlloch M (2010) K+ starvation inhibits water-stress-induced stomatal closure via ethylene synthesis in sunflower plants. Journal of Experimental Botany 61, 1139–1145.
K+ starvation inhibits water-stress-induced stomatal closure via ethylene synthesis in sunflower plants.Crossref | GoogleScholarGoogle Scholar | 20054030PubMed |

Besson-Bard A, Pugin A, Wendehenne D (2008) New insights into nitric oxide signaling in plants. Annual Review of Plant Biology 59, 21–39.
New insights into nitric oxide signaling in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXntFaqs7Y%3D&md5=4053b9eba250cf01703a80c0b4d71b25CAS | 18031216PubMed |

Beyer EMJ (1976) A potent inhibitor of ethylene action in plants. Plant Physiology 58, 268–271.
A potent inhibitor of ethylene action in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE28XlvVOks7Y%3D&md5=26356776ddb2fb5e387bd6d2cc6595ecCAS | 16659660PubMed |

Crawford NM (2006) Mechanisms for nitric oxide synthesis in plants. Journal of Experimental Botany 57, 471–478.
Mechanisms for nitric oxide synthesis in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XovVCrug%3D%3D&md5=6878df2c4d81ef4747ed5136faf3ea99CAS | 16356941PubMed |

Desikan R, Last K, Harrett-Williams R, Tagliavia C, Harter K, Hooley R, Hancock JT, Neill SJ (2006) Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis. The Plant Journal 47, 907–916.
Ethylene-induced stomatal closure in Arabidopsis occurs via AtrbohF-mediated hydrogen peroxide synthesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtVylsL%2FP&md5=e446d7784fa0d158d6eddf075d0c345bCAS | 16961732PubMed |

Eisinger WR, Swartz RE, Bogomolni RA, Taiz L (2000) The ultraviolet action spectrum for stomatal opening in broad bean. Plant Physiology 122, 99–106.
The ultraviolet action spectrum for stomatal opening in broad bean.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXmvFanug%3D%3D&md5=6c2a168ab8c93405443e78b9b96dcadbCAS | 10631253PubMed |

Garcia-Mata C, Lamattina L (2001) Nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress. Plant Physiology 126, 1196–1204.
Nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3Mvgs1GksA%3D%3D&md5=40e0d71881827c8681ab15863387a530CAS | 11457969PubMed |

Garty J, Weissman L, Levin T, Garty-Spitz R, Lehr H (2004) Impact of UV-B, heat and chemicals on ethylene-production of lichens. Journal of Atmospheric Chemistry 49, 251–266.
Impact of UV-B, heat and chemicals on ethylene-production of lichens.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhslemur4%3D&md5=34d3ee4942c84b4ffb48b4177329550aCAS |

Gitz DC, Liu-Gitz L (2003) How do UV photomorphogenic responses confer water stress tolerance? Photochemistry and Photobiology 78, 529–534.
How do UV photomorphogenic responses confer water stress tolerance?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXhsler&md5=3409ba31ef7e467c479ef69bd8432f4bCAS | 14743860PubMed |

Gunderson CA, Taylor GE (1991) Ethylene directly inhibits foliar gas exchange in Glycine max. Plant Physiology 95, 337–339.
Ethylene directly inhibits foliar gas exchange in Glycine max.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXnvVymtA%3D%3D&md5=e874144474f63b5d2cb7ceb7ca4377ceCAS | 16667976PubMed |

Guo H, Ecker JR (2004) The ethylene signalling pathway: new insights. Current Opinion in Plant Biology 7, 40–49.
The ethylene signalling pathway: new insights.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltFWqtg%3D%3D&md5=695b9893331898db04f58906394bc490CAS | 14732440PubMed |

Guo FQ, Okamoto M, Crawford NM (2003) Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302, 100–103.
Identification of a plant nitric oxide synthase gene involved in hormonal signaling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXnslSjs7o%3D&md5=86dc8811cb42b4b35676fc0fd3787770CAS | 14526079PubMed |

He J-M, She X-P, Liu C, Zhao W-M (2004) Stomatal and nonstomatal limitations of photosynthesis in mung bean leaves under the combination of enhanced UV-B radiation and NaCl stress. Journal of Plant Physiology and Molecular Biology 30, 53–58.

He J-M, Xu H, She X-P, Song X-G, Zhao W-M (2005) The role and the interrelationship of hydrogen peroxide and nitric oxide in the UV-B-induced stomatal closure in broad bean. Functional Plant Biology 32, 237–247.
The role and the interrelationship of hydrogen peroxide and nitric oxide in the UV-B-induced stomatal closure in broad bean.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXivFWjt74%3D&md5=5b209944cd6ae9660e01367112ca97eeCAS |

He J-M, Bai X-L, Wang R-B, Cao B, She X-P (2007) The involvement of nitric oxide in ultraviolet-B-inhibited pollen germination and tube growth of Paulownia tomentosa in vitro. Physiologia Plantarum 131, 273–282.

Jansen MAK, Noort REVD (2000) Ultraviolet-B radiation induces complex alterations in stomatal behaviour. Physiologia Plantarum 110, 189–194.
Ultraviolet-B radiation induces complex alterations in stomatal behaviour.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXnt1Cntb4%3D&md5=2f13f934a0a8b48bda1e03fe322e0d7bCAS |

Kojima H, Nakatsubo N, Kikuchi K, Urano Y, Higuchi T, Tanaka J, Kudo Y, Nagano T (1998) Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF-2 DA. Neuroreport 9, 3345–3348.
Direct evidence of NO production in rat hippocampus and cortex using a new fluorescent indicator: DAF-2 DA.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXotV2ktrc%3D&md5=f5c7ec0e1d967d0f3c09cd888e16d8cbCAS | 9855277PubMed |

Kolla VA, Raghavendra AS (2007) Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum. Physiologia Plantarum 130, 91–98.
Nitric oxide is a signaling intermediate during bicarbonate-induced stomatal closure in Pisum sativum.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXltFCqtrg%3D&md5=524dab4ab5d1f79a554f90f1402fbf60CAS |

Levitt LK, Stein DB, Rubbinstein B (1987) Promotion of stomatal opening by indoleacetic acid and ethrel in epidermal strips of Vicia faba L. Plant Physiology 85, 318–321.
Promotion of stomatal opening by indoleacetic acid and ethrel in epidermal strips of Vicia faba L.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1cXjs1Sr&md5=3959453e46328374463d5367f3790618CAS | 16665694PubMed |

Li J, Qiu LY, Zhao FG, Hou LX, Liu X (2007) The role of nitric oxide in ethylene-induced stomatal closure in Vicia faba L. Journal of Plant Physiology and Molecular Biology 33, 349–353.

Liu X, Zhang SQ, Lou CH (2003) Involvement of nitric oxide in the signal transduction of salicylic acid regulating stomatal movement. Chinese Science Bulletin 48, 449–452.

Liu X, Shi WL, Zhang SQ, Lou CH (2005) Nitric oxide involved in signal transduction of jasmonic acid-induced stomatal closure of Vicia faba L. Chinese Science Bulletin 50, 520–525.

Liu GH, Liu J, Hou LX, Tang J, Liu X (2009) No may function in the downstream of Ca2+ in ethylene induced stomatal closure in Vicia faba L. Journal of Molecular Cell Biology 42, 145–154.

Liu J, Liu GH, Hou LX, Liu X (2010) Ethylene-induced nitric oxide production and stomatal closure in Arabidopsis thaliana depending on changes in cytosolic pH. Chinese Science Bulletin 55, 2403–2409.
Ethylene-induced nitric oxide production and stomatal closure in Arabidopsis thaliana depending on changes in cytosolic pH.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXpvFensLg%3D&md5=16079b8321e2a0e4666ca6c9a4fbbf34CAS |

Mackerness SAH, John CF, Jordan B, Thomas B (2001) Early signaling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide. FEBS Letters 489, 237–242.
Early signaling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXpsF2nuw%3D%3D&md5=4cd5c06ae405fbfa40c9f5f1d7529006CAS | 11165257PubMed |

Madhavan S, Chrmoinski A, Smith BN (1983) Effect of ethylene on stomatal opening in tomato and carnation leaves. Plant & Cell Physiology 24, 569–572.

Merritt F, Kemper A, Tallman G (2001) Inhibitors of ethylene biosynthesis inhibit auxin-induced stomatal opening in epidermis detached from leaves of Vicia faba L. Plant & Cell Physiology 42, 223–230.
Inhibitors of ethylene biosynthesis inhibit auxin-induced stomatal opening in epidermis detached from leaves of Vicia faba L.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXhsFynu7k%3D&md5=61f7ed34363ef53c8ae40f4cdc3c55ecCAS | 11230577PubMed |

Moreau M, Lee GI, Wang Y, Crane BR, Klessig DF (2008) AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase. Journal of Biological Chemistry 283, 32 957–32 967.
AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtlOjtLbO&md5=de5ae710f4ba3decde0e11abfe357e09CAS |

Musil CF, Wand SJE (1993) Responses of sclerophyllous Ericaceae to enhanced level of ultraviolet-B radiation. Environmental and Experimental Botany 33, 233–242.
Responses of sclerophyllous Ericaceae to enhanced level of ultraviolet-B radiation.Crossref | GoogleScholarGoogle Scholar |

Nara A, Takeuchl Y (2002) Ethylene evolution from tobacco leaves irradiated with UV-B. Journal of Plant Research 115, 247–253.
Ethylene evolution from tobacco leaves irradiated with UV-B.Crossref | GoogleScholarGoogle Scholar | 12582726PubMed |

Neill S, Barros R, Bright J, Desikan R, Hancock J, Harrison J, Morris P, Ribeiro D, Wilson I (2008) Nitric oxide, stomatal closure, and abiotic stress. Journal of Experimental Botany 59, 165–176.
Nitric oxide, stomatal closure, and abiotic stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXjsVamt70%3D&md5=ea1a69b5b8c69beea49a61cf1e5261e6CAS | 18332225PubMed |

Nogués S, Allen DJ, Morison JIL, Baker NR (1999) Characterization of stomatal closure caused by ultraviolet-B radiation. Plant Physiology 121, 489–496.
Characterization of stomatal closure caused by ultraviolet-B radiation.Crossref | GoogleScholarGoogle Scholar | 10517840PubMed |

Pallas JE, Kays SJ (1982) Inhibition of photosynthesis by ethylene – a stomatal effect. Plant Physiology 70, 598–601.
Inhibition of photosynthesis by ethylene – a stomatal effect.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL38XlsF2ksLs%3D&md5=a4458b96b8671248a14128e394f77f67CAS | 16662540PubMed |

Qiao W, Fan LM (2008) Nitric oxide signaling in plant responses to abiotic stresses. Journal of Integrative Plant Biology 50, 1238–1246.
Nitric oxide signaling in plant responses to abiotic stresses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtlaitbrF&md5=54421fdf93aa79a26f1d3597f7ec6e7dCAS | 19017111PubMed |

Ramani S, Chelliah J (2007) UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures. BMC Plant Biology 7, 61–77.
UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures.Crossref | GoogleScholarGoogle Scholar | 17988378PubMed |

Rozema J, Boelen P, Blokker P (2005) Depletion of stratospheric ozone over the Antarctic and Arctic: responses of plants of polar terrestrial ecosystems to enhanced UV-B, an overview. Environmental Pollution 137, 428–442.
Depletion of stratospheric ozone over the Antarctic and Arctic: responses of plants of polar terrestrial ecosystems to enhanced UV-B, an overview.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXlvFygurw%3D&md5=f85f273d023f9d86e6dee98bb6274326CAS | 16005756PubMed |

Sisler EC, Serek M (1997) Inhibitors of ethylene responses in plants at the receptor level: recent developments. Physiologia Plantarum 100, 577–582.
Inhibitors of ethylene responses in plants at the receptor level: recent developments.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXks1yqs7k%3D&md5=8e3b2897dc335db17b7ba88732dfb0d9CAS |

Tamaoki M, Matsuyama T, Kanna M, Nakajima N, Kubo A, Aono M, Saji H (2003) Differential ozone sensitivity among Arabidopsis accessions and its relevance to ethylene synthesis. Planta 216, 552–560.

Tanaka Y, Sano T, Tamaoki M, Nakajima N, Kondo N, Hasezawa S (2005) Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis. Plant Physiology 138, 2337–2343.
Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXps12ltr0%3D&md5=1cae121eb88d8f15b1e45c4c7233b7ceCAS | 16024687PubMed |

Tanaka Y, Sano T, Tamaoki M, Nakajima N, Kondo N, Hasezawa S (2006) Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis. Journal of Experimental Botany 57, 2259–2266.
Cytokinin and auxin inhibit abscisic acid-induced stomatal closure by enhancing ethylene production in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XnvF2mu70%3D&md5=54cffbf01030e01012e83853837a1b1dCAS | 16798847PubMed |

Tissera P, Ayres PG (1986) Endogenous ethylene affects the behavior of stomata in epidermis isolated from rust infected faba bean (Vicia faba L.). New Phytologist 104, 53–61.
Endogenous ethylene affects the behavior of stomata in epidermis isolated from rust infected faba bean (Vicia faba L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL28XlvVOrsL8%3D&md5=dfd0070aad57d3ff56c7a5abf2c64e15CAS |

Tossi V, Lamattina L, Cassia R (2009) An increase in the concentration of abscisic acid is critical for nitric oxide-mediated plant adaptive responses to UV-B irradiation. New Phytologist 181, 871–879.
An increase in the concentration of abscisic acid is critical for nitric oxide-mediated plant adaptive responses to UV-B irradiation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXjsFGiur8%3D&md5=c2d6be3611f77612f3d396647805f741CAS | 19140950PubMed |

Wang KLC, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. The Plant Cell 14, 131–151.

Wang Y, Feng H, Qu Y, Cheng J, Zhao Z, Zhang M, Wang X, An L (2006) The relationship between reactive oxygen species and nitric oxide in ultraviolet-B-induced ethylene production in leaves of maize seedlings. Environmental and Experimental Botany 57, 51–61.
The relationship between reactive oxygen species and nitric oxide in ultraviolet-B-induced ethylene production in leaves of maize seedlings.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XkslSks7o%3D&md5=0323bfdc9a4dbf6f183d948952b0ae43CAS |

Wilkinson S, Davies WJ (2009) Ozone suppresses soil drying- and abscisic acid (ABA)-induced stomatal closure via an ethylene-dependent mechanism. Plant, Cell & Environment 32, 949–959.
Ozone suppresses soil drying- and abscisic acid (ABA)-induced stomatal closure via an ethylene-dependent mechanism.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtValtbnL&md5=b754d6b1ed10000de069603a1061082bCAS | 19302171PubMed |

Wilkinson S, Davies WJ (2010) Drought, ozone, ABA and ethylene: new insights from cell to plant to community. Plant, Cell & Environment 33, 510–525.
Drought, ozone, ABA and ethylene: new insights from cell to plant to community.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXltV2hurs%3D&md5=7ff6bcfef3fea72c38cb0ad40ab89c05CAS | 19843256PubMed |

Wilson ID, Neill SJ, Hancock JT (2008) Nitric oxide synthesis and signalling in plants. Plant, Cell & Environment 31, 622–631.
Nitric oxide synthesis and signalling in plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXlvFehur4%3D&md5=0ae61fe5646fbe5fd83f130195d569c2CAS | 18034772PubMed |

Young TE, Meeley RB, Gallie DR (2004) ACC synthase expression regulates leaf performance and drought tolerance in maize. The Plant Journal 40, 813–825.
ACC synthase expression regulates leaf performance and drought tolerance in maize.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjvFWgtQ%3D%3D&md5=0a5bf8738f3e3734ce23ed59340a7cc3CAS | 15546363PubMed |