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RESEARCH ARTICLE

Proteomic prospects for tolerance of sunflower (Helianthus annuus) to drought stress during the flowering stage

Mehdi Ghaffari A C , Mahmoud Toorchi B , Mostafa Valizadeh B and Mohammadreza Shakiba B
+ Author Affiliations
- Author Affiliations

A Seed and Plant Improvement Institute, Agricultural Research Education and Extension Organisation (AREEO), Karaj, Iran.

B Department of Crop Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz 51666-16471, Iran.

C Corresponding author. Email: ghaffari@areeo.ac.ir

Crop and Pasture Science 68(5) 457-465 https://doi.org/10.1071/CP17105
Submitted: 28 February 2017  Accepted: 15 May 2017   Published: 19 June 2017

Abstract

In order to understanding proteomic basis of drought tolerance in sunflower (Helianthus annuus L.), two contrasting inbred lines were subjected to drought stress during the flowering stage for two years. Proteins were extracted from leaves of well-watered and drought-treated plants by using the TCA–acetone precipitation method and analysed by two-dimensional polyacrylamide gel electrophoresis followed by nanoscale liquid chromatography coupled to tandem mass spectrometry for identification of affected proteins. When comparing proteomic patterns, 18 proteins were changed by drought stress in sensitive lines and 24 proteins in tolerant lines. Concurrent down-expressions of oxygen-evolving enhancer and ferredoxin-NADP reductase were considered as primary drought sensors that mediate downstream pathways to cope with drought conditions. Differential and line-specific proteomic changes were attributed as the source for contrasting response to drought stress. According to the results, scavenging of reactive oxygen species, conservation of energy and water, and cell-structure integrity constituted the major aspects of drought tolerance in sunflower.

Additional keywords: drought resistance, nano-LC–MS/MS, photosynthesis, proteomics, ROS, 2-DE.


References

Ali GM, Komatsu S (2006) Proteomic analysis of rice leaf sheath during drought stress. Journal of Proteome Research 5, 396–403.
Proteomic analysis of rice leaf sheath during drought stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XlvFSh&md5=3a64476d372c21b981e2d09aa8273f4bCAS |

Bagniewska-Zadworna A (2008) The root microtubule cytoskeleton and cell cycle analysis through desiccation of Brassica napus seedlings. Protoplasma 233, 177–185.
The root microtubule cytoskeleton and cell cycle analysis through desiccation of Brassica napus seedlings.Crossref | GoogleScholarGoogle Scholar |

Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, Dirkse W, Van Staveren M, Stiekema W, Drost L, Ridley P, Hudson SA, Patel K, Murphy G, Piffanelli P, Wedler H, Wedler E, Wambutt R, Weitzenegger T, Pohl TM, Terryn N, Gielen J, Villarroel R, De Clerck R, Van Montagu M, Lecharny A, Auborg S, Gy I, Kreis M, Lao N, Kavanagh T, Hempel S, Kotter P, Entian KD, Rieger M, Schaeffer M, Funk B, Mueller-Auer S, Silvey M, James RMA, Pons A, Puigdomenech P, Douka A, Voukelatou E, Milioni D, Hatzopoulos P, Piravandi E, Obermaier B, Hilbert H, Dusterhoft A, Moores T, Jones JDG, Eneva T, Palme K, Benes V, Rechman S, Ansorge W, Cooke R, Berger C, Delseny M, Voet M, Volckaert G, Mewes HW, Klosterman S, Schueller C, Chalwatzis N (1998) Analysis of 1.9Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391, 485–488.

Bhushan D, Pandey A, Choudhary MK, Datta A, Chakraborty S, Chakraborty N (2007) Comparative proteomics analysis of differentially expressed proteins in chickpea extracellular matrix during dehydration stress. Molecular & Cellular Proteomics 6, 1868–1884.
Comparative proteomics analysis of differentially expressed proteins in chickpea extracellular matrix during dehydration stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsVeisbrL&md5=b4970a38cf42873495f12a83fa80e517CAS |

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE28XksVehtrY%3D&md5=ec1c81995b5f1a7887c0acb71f446495CAS |

Bray EA (1997) Plant responses to water deficit. Trends in Plant Science 2, 48–54.
Plant responses to water deficit.Crossref | GoogleScholarGoogle Scholar |

Caruso G, Cavaliere C, Foglia P, Gubbiotti R, Samperi R, Laganà A (2009) Analysis of drought responsive proteins in wheat (Triticum durum) by 2D-PAGE and MALDI-TOF mass spectrometry. Plant Science 177, 570–576.
Analysis of drought responsive proteins in wheat (Triticum durum) by 2D-PAGE and MALDI-TOF mass spectrometry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXht1OrurvI&md5=4c603ba39ebf784dc79a8b3b48a5ba54CAS |

Castillejo MA, Maldonado AM, Ogueta S, Jorrin JV (2008) Proteomic analysis of responses to drought stress in sunflower (Helianthus annuus L.) leaves by 2DE gel electrophoresis and mass spectrometry. The Open Proteomics Journal 1, 59–71.
Proteomic analysis of responses to drought stress in sunflower (Helianthus annuus L.) leaves by 2DE gel electrophoresis and mass spectrometry.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXps1Wgsrc%3D&md5=091f0a5a70a3d3ddba4c9ecc0126c9d8CAS |

Cattivelli L, Rizza F, Badeck FW, Mazzucotelli E, Mastrangelo AM, Francia E, Mare C, Tondelli A, Stanca AM (2008) Drought tolerance improvement in crop plants: An integrative view from breeding to genomics. Field Crops Research 105, 1–14.
Drought tolerance improvement in crop plants: An integrative view from breeding to genomics.Crossref | GoogleScholarGoogle Scholar |

Chaves MM, Maroco JP, Pereira JS (2003) Understanding plant responses to drought from genes to the whole plant. Functional Plant Biology 30, 239–264.
Understanding plant responses to drought from genes to the whole plant.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjtVKlt7o%3D&md5=d4de6ee5dbb0188566e48cc6f7ddae43CAS |

Chimenti CA, Pearson J, Hall AJ (2002) Osmotic adjustment and yield maintenance under drought in sunflower. Field Crops Research 75, 235–246.
Osmotic adjustment and yield maintenance under drought in sunflower.Crossref | GoogleScholarGoogle Scholar |

Fang X, Ma H, Lu D, Yu H, Lai W, Ruan S (2011) Comparative proteomics analysis of proteins expressed in the I-1 and I-2 internodes of strawberry stolons. Proteome Science 9, 1–15.

Fulda S, Mikkat S, Stegmann H, Horn R (2011) Physiology and proteomics of drought stress acclimation in sunflower (Helianthus annuus L.). Plant Biology 13, 632–642.
Physiology and proteomics of drought stress acclimation in sunflower (Helianthus annuus L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXot1Ogu7o%3D&md5=b209dd7d8f37237044306f5324b2ac49CAS |

Garcia JS, Souza GHMF, Eberlin MN, Arruda MAZ (2009) Evaluation of metal-ion stress in sunflower (Helianthus annuus L.) leaves through proteomic changes. Metallomics 1, 107–113.
Evaluation of metal-ion stress in sunflower (Helianthus annuus L.) leaves through proteomic changes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXht1Srtrs%3D&md5=a459e77084e168aa04ad083c4c9cd3c0CAS |

Ghaffari M, Toorchi M, Valizadeh M, Shakiba MR (2012) Morpho-physiological screening of sunflower inbred lines under drought stress condition. Turkish Journal of Field Crops 17, 185–190.

Ghaffari M, Toorchi M, Valizadeh M, Komatsu S (2013) Differential response of root proteome to drought stress in drought sensitive and tolerant sunflower inbred lines. Functional Plant Biology 40, 609–617.
Differential response of root proteome to drought stress in drought sensitive and tolerant sunflower inbred lines.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXpsVOksrg%3D&md5=f0dde82f77fc9f2801909a54ff5ea9d6CAS |

Giardi MT, Cona A, Geiken B, Kucera T, Masojidek J, Mattoo AK (1996) Long-term drought stress induces structural and functional reorganization of photosystem II. Planta 199, 118–125.
Long-term drought stress induces structural and functional reorganization of photosystem II.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XisVens7o%3D&md5=56b986a94c57ffa8bca36e72f735e659CAS |

Guo P, Baum M, Grando S, Ceccarelli S, Bai G, Li R, von Korff M, Varshney RK, Graner A, Valkoun J (2009) Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage. Journal of Experimental Botany 60, 3531–3544.
Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtVWitr3J&md5=8ba879d5180b626dcfd40a8465393d64CAS |

Hajheidari M, Abdollahian-Noghabi M, Askari H, Heidari M, Sadeghian SY, Ober ES, Salekdeh GH (2005) Proteome analysis of sugar beet leaves under drought stress. Proteomics 5, 950–960.
Proteome analysis of sugar beet leaves under drought stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXivVGgtrg%3D&md5=ce9f5b652e9a7ee783f357fe67716467CAS |

Hashiguchi A, Ahsan N, Komatsu S (2010) Proteomics application of crops in the context of climatic changes. Food Research International 43, 1803–1813.
Proteomics application of crops in the context of climatic changes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtFCkt7vK&md5=abda6b5f5380624da05443a4c048b97aCAS |

Havaux M, Canaani O, Malkin S (1987) Inhibition of photosynthetic activities under slow water stress measured in vivo by the photo acoustic method. Physiologia Plantarum 70, 503–510.
Inhibition of photosynthetic activities under slow water stress measured in vivo by the photo acoustic method.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2sXltlWitrs%3D&md5=4c4e6675d8d21e1ba74959961d2259b8CAS |

Huber SC, Akazawa T (1986) A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. Plant Physiology 81, 1008–1013.
A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL28XltlSkt74%3D&md5=adc54a0251710980d2d9fa90c02209c7CAS |

Human JJ, Dutoit D, Bezuidenhout HD, Bruyn LP (1990) The influence of plant water stress on net photosynthesis and yield of sunflower. Crop Science 164, 231–241.
The influence of plant water stress on net photosynthesis and yield of sunflower.Crossref | GoogleScholarGoogle Scholar |

Jacobs DI, van der Heijden R, Verpoorte R (2000) Proteomics in plant biotechnology and secondary metabolism research. Phytochemical Analysis 11, 277–287.
Proteomics in plant biotechnology and secondary metabolism research.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXntVSlurk%3D&md5=05324936fcdeb6273d263279451462e4CAS |

Kamal AHM, Kim KH, Shin KH, Choi JS, Baik BK, Tsujimoto H, Heo HY, Park CS, Woo SH (2010) Abiotic stress responsive proteins of wheat grain determined using proteomics technique. Australian Journal of Crop Science 4, 196–208.

Kausar R, Arshad M, Shahzad A, Komatsu S (2013) Proteomics analysis of sensitive and tolerant barley genotypes under drought stress. Amino Acids 44, 345–359.
Proteomics analysis of sensitive and tolerant barley genotypes under drought stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3sXhtFyitL0%3D&md5=e77a674f2144978bdb21e16079aa7deaCAS |

Komatsu S, Konishi H, Hashimoto M (2007) The proteomics of plant cell membranes. Journal of Experimental Botany 58, 1103–1112.

Kottapalli KR, Rakwal R, Shibato J, Burow G, Tissue D, Burke J, Puppala N, Burow M, Payton P (2009) Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes. Plant, Cell & Environment 32, 380–407.
Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXkslKjsLo%3D&md5=9957685886b6251d77aa10423dc0da1fCAS |

Laporte MM, Shen B, Tarczynski MC (2002) Engineering for drought avoidance: Expression of maize NADP malic enzyme in tobacco results in altered stomatal function. Journal of Experimental Botany 53, 699–705.
Engineering for drought avoidance: Expression of maize NADP malic enzyme in tobacco results in altered stomatal function.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XitlCks7k%3D&md5=8cc3eb841da018d797890d51ef24f665CAS |

Lawlor DW, Cornic G (2002) Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants. Plant, Cell & Environment 25, 275–294.
Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in higher plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xhslakur0%3D&md5=82115f2e5fc3c0cf3e69e9e93e94fa67CAS |

Lee S, Lee EJ, Yang EJ, Lee JE, Park AR, Song WH, Park OK (2004) Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. The Plant Cell 16, 1378–1391.
Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXlsFWlsL4%3D&md5=401b008b009ded466ddfb8cd7b2630e4CAS |

Lopes Júnior CA, Barbosa HS, Galazzi RM, Koolen HHF, Gozzo FC, Arruda MAZ (2015) Evaluation of proteome alterations induced by cadmium stress in sunflower (Helianthus annuus L.) cultures. Ecotoxicology and Environmental Safety 119, 170–177.
Evaluation of proteome alterations induced by cadmium stress in sunflower (Helianthus annuus L.) cultures.Crossref | GoogleScholarGoogle Scholar |

Merewitz EB, Gianfagna T, Huang B (2011) Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis. Journal of Experimental Botany 62, 5311–5333.
Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhsFCit73O&md5=149ba8b1e29cc5fb59172868ed2a8395CAS |

Mohammadi PP, Moieni A, Komatsu S (2012) Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress. Amino Acids 43, 2137–2152.
Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC38XhsFSqtbnJ&md5=a38812d1841e3f79aa86ec0a1cf308f5CAS |

Mohayeji M, Capriotti AL, Cavaliere C, Piovesana S, Samperi R, Stampachiacchiere S, Toorchi M, Lagana A (2014) Heterosis profile of sunflower leaves: a label free proteomics approach. Journal of Proteomics 99, 101–110.
Heterosis profile of sunflower leaves: a label free proteomics approach.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC2cXjsF2hurw%3D&md5=26aa3d3e970858095a18e03adf1f82f1CAS |

Rauf S (2008) Breeding sunflower (Helianthus annuus L.) for drought tolerance. Communications in Biometry and Crop Science 3, 29–44.

Reddy GKM, Dangi KS, Kumar SS, Reddy AV (2003) Effect of moisture stress on seed yield and quality in sunflower, Helianthus annuus L. Journal of Oilseeds Research 20, 282–283.

Rodriguez RE, Lodeyro A, Poli HO, Zurbriggen M, Peisker M, Palatnik JF, Tognetti VB, Tschiersch H, Hajirezaei MR, Valle EM, Carrillo N (2007) Transgenic tobacco plants overexpressing chloroplastic FNR display normal rates of photosynthesis and increased tolerance to oxidative stress. Plant Physiology 143, 639–649.
Transgenic tobacco plants overexpressing chloroplastic FNR display normal rates of photosynthesis and increased tolerance to oxidative stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhvFWnsLk%3D&md5=86fba35d72ca4e972fb1456ee6e9ca95CAS |

Rossignol M, Peltier JB, Mock HP, Matros A, Maldonado AM, Jorrín JV (2006) Plant proteome analysis: a 2004–2006 update. Proteomics 6, 5529–5548.
Plant proteome analysis: a 2004–2006 update.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtFyms7bE&md5=a17927845b8b92139e0e6ae40d699be4CAS |

Salekdeh GH, Siopongco J, Wade LJ, Ghareyazie B, Bennett J (2002) Proteomic analysis of rice leaves during drought stress and recovery. Proteomics 2, 1131–1145.
Proteomic analysis of rice leaves during drought stress and recovery.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38Xnsl2msbo%3D&md5=b3927e5fd56a8464c083e617a5c14f80CAS |

Schneiter AA, Miller JF (1981) Description of sunflower growth stages. Crop Science 21, 901–903.
Description of sunflower growth stages.Crossref | GoogleScholarGoogle Scholar |

Sharp RE, Boyer JS (1986) Photosynthesis at low water potentials in sunflower: Lack of photoinhibitory effects. Plant Physiology 82, 90–95.
Photosynthesis at low water potentials in sunflower: Lack of photoinhibitory effects.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL28Xls12itL0%3D&md5=ca8f8aeaef757ca89c8ed96c838ffcb1CAS |

Sionit N, Kramer PJ (1976) Water potential and stomatal resistance of sunflower and soybean subjected to water stress during various growth stages. Plant Physiology 58, 537–540.
Water potential and stomatal resistance of sunflower and soybean subjected to water stress during various growth stages.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3cnhtFyqsQ%3D%3D&md5=e1aba76cc1d51a88c25d0e3ce0721714CAS |

Sulmon C, Gouesbet G, Amrani EA, Couee I (2006) Sugar-induced tolerance to the herbicide atrazine in Arabidopsis seedlings involves activation of oxidative and xenobiotic stress responses. Plant Cell Reports 25, 489–498.
Sugar-induced tolerance to the herbicide atrazine in Arabidopsis seedlings involves activation of oxidative and xenobiotic stress responses.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xjs1Kksrc%3D&md5=899a84507fbb8b49454be261b2c2535fCAS |

Taiz L, Zeiger E (2002) ‘Plant physiology.’ 3rd edn (Sinauer Associates: Sunderland, MA, USA)

Takeuchi Y, Akagi H, Kamasawa N, Osumi M, Honda H (2000) Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme. Planta 211, 265–274.
Aberrant chloroplasts in transgenic rice plants expressing a high level of maize NADP-dependent malic enzyme.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXksFGhtb8%3D&md5=b3385f1176915156910e4d7231c8b96cCAS |

Vander Willigen C, Pammenter NW, Jaffer MA, Mundree SG, Farrant JM (2003) An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccation-tolerant and -sensitive tissues. Functional Plant Biology 30, 281–290.
An ultrastructural study using anhydrous fixation of Eragrostis nindensis, a resurrection grass with both desiccation-tolerant and -sensitive tissues.Crossref | GoogleScholarGoogle Scholar |

Vinocur B, Altman A (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Current Opinion in Biotechnology 16, 123–132.
Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXjtlamu74%3D&md5=2640f9288206b026d63e21ea92785a95CAS |

Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat shock proteins and molecular chaperones in the abiotic stress response. Trends in Plant Science 9, 244–252.
Role of plant heat shock proteins and molecular chaperones in the abiotic stress response.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXjvVemtbw%3D&md5=ed5465b20c35e2e3531600fd1eb4b83bCAS |

Wang X, Chen S, Zhang H, Shi L, Cao F, Guo L, Xie Y, Wang T, Yan X, Dai S (2010) Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis. Journal of Proteome Research 9, 6561–6577.
Desiccation tolerance mechanism in resurrection fern-ally Selaginella tamariscina revealed by physiological and proteomic analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtlynsbnK&md5=ca5c709c2d92ba51e22bec9b7e687accCAS |

Ward JH (1963) Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association 58, 236–244.
Hierarchical grouping to optimize an objective function.Crossref | GoogleScholarGoogle Scholar |

Xiao X, Yang F, Zhang S, Korpelainen H, Li C (2009) Physiological and proteomic responses of two contrasting Populus cathayana populations to drought stress. Physiologia Plantarum 136, 150–168.
Physiological and proteomic responses of two contrasting Populus cathayana populations to drought stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXntlehsLY%3D&md5=c48adbb34da561adc429abbad13bdde9CAS |

Yan S, Tang Z, Su W, Sun W (2005) Proteomic analysis of salt stress-responsive proteins in rice root. Proteomics 5, 235–244.
Proteomic analysis of salt stress-responsive proteins in rice root.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhtleisL8%3D&md5=037713803c53be9417cf61976cb0b540CAS |