Functional Plant Biology Functional Plant Biology Society
Plant function and evolutionary biology
RESEARCH ARTICLE

Constitutive expression of CaHSP22.5 enhances chilling tolerance in transgenic tobacco by promoting the activity of antioxidative enzymes

Meifang Li A , Lusha Ji A , Zefeng Jia A , Xinghong Yang B , Qingwei Meng B and Shangjing Guo A C
+ Author Affiliations
- Author Affiliations

A College of Life Science, Liaocheng University, Liaocheng 252000, China.

B State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271018, China.

C Corresponding author. Email: guoshangjing@lcu.edu.cn

Functional Plant Biology - https://doi.org/10.1071/FP17226
Submitted: 10 August 2017  Accepted: 29 November 2017   Published online: 5 January 2018

Abstract

Chilling stress limits the productivity and geographical distribution of many organisms throughout the world. In plants, the small heat shock proteins (sHSPs) belong to a group of proteins known as chaperones. The sweet pepper (Capsicum annuum L.) cDNA clone CaHSP22.5, which encodes an endoplasmic reticulum-located sHSP (ER-sHSP), was isolated and introduced into tobacco (Nicotiana tabacum L.) plants and Escherichia coli. The performance index and the maximal efficiency of PSII photochemistry (Fv/Fm) were higher and the accumulation of H2O2 and superoxide radicals (O2) was lower in the transgenic lines than in the untransformed plants under chilling stress, which suggested that CaHSP22.5 accumulation enhanced photochemical activity and oxidation resistance. However, purified CaHSP22.5 could not directly reduce the contents of H2O2 and O2 in vitro. Additionally, heterologously expressed recombinant CaHSP22.5 enhanced E. coli viability under oxidative stress, helping to elucidate the cellular antioxidant function of CaHSP22.5 in vivo. At the same time, antioxidant enzyme activity was higher, which was consistent with the lower relative electrolyte conductivity and malondialdehyde contents of the transgenic lines compared with the wild-type. Furthermore, constitutive expression of CaHSP22.5 decreased the expression of other endoplasmic reticulum molecular chaperones, which indicated that the constitutive expression of ER-sHSP alleviated endoplasmic reticulum stress caused by chilling stress in plants. We hypothesise that CaHSP22.5 stabilises unfolded proteins as a chaperone and increases the activity of reactive oxygen species-scavenging enzymes to avoid oxidation damage under chilling stress, thereby suggesting that CaHSP22.5 could be useful for improving the tolerance of chilling-sensitive plant types.

Additional keywords: ER-sHSP, CaHSP22.5, Chill tolerance, Antioxidative enzyme.


References

Aghdam MS, Sevillano L, Flores FB, Bodbodak S (2013) Heat shock proteins as biochemical markers for postharvest chilling stress in fruits and vegetables. Scientia Horticulturae 160, 54–64.
Heat shock proteins as biochemical markers for postharvest chilling stress in fruits and vegetables.CrossRef | 1:CAS:528:DC%2BC3sXht1elu7fL&md5=0db1b585c0b2fcfcfb67aa748334ff9aCAS |

Alvim FC, Carolino SM, Cascardo JC, Nunes CC (2001) Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress. Plant Physiology 126, 1042–1054.
Enhanced accumulation of BiP in transgenic plants confers tolerance to water stress.CrossRef | 1:CAS:528:DC%2BD3MXlsVarur4%3D&md5=8a802b862f19c1312c32aae9e1e7affaCAS |

Baldwin AJ, Hilton GR, Lioe H, Bagneris C, Benesch JL, Kay LE (2011) Quaternary dynamics of aB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus. Journal of Molecular Biology 413, 310–320.
Quaternary dynamics of aB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus.CrossRef | 1:CAS:528:DC%2BC3MXhtlaksb7P&md5=e77b9266368da9a8e04be1427cd5d0cdCAS |

Bartoli CG, Simontacchi M, Tambussi E, Beltrano J, Montaldi E, Puntarulo S (1999) Drought and watering-dependent oxidative stress: effect on anti-oxidant content in Triticum aestivum L. leaves. Journal of Experimental Botany 50, 375–383.
Drought and watering-dependent oxidative stress: effect on anti-oxidant content in Triticum aestivum L. leaves.CrossRef | 1:CAS:528:DyaK1MXhvFyitrg%3D&md5=08fbc1d0d2471334c26a0589938c3ca3CAS |

Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.
A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein–dye binding.CrossRef | 1:CAS:528:DyaE28XksVehtrY%3D&md5=90b0b14534fb4b0d25711d896ae5505eCAS |

Cai GH, Wang GD, Wang L, Pan JW, Liu Y, Li DQ (2014) ZmMKK1, a novel group A mitogen-activated protein kinase kinase gene in maize, conferred chilling stress tolerance and was involved in pathogen defense in transgenic tobacco. Plant Science 214, 57–73.
ZmMKK1, a novel group A mitogen-activated protein kinase kinase gene in maize, conferred chilling stress tolerance and was involved in pathogen defense in transgenic tobacco.CrossRef | 1:CAS:528:DC%2BC3sXhvVWksrnP&md5=c2ec1ac3c59309713aab4753d9af0361CAS |

Elstner FF, Heupel C (1976) Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase. Analytical Biochemistry 70, 616–620.
Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase.CrossRef | 1:CAS:528:DyaE28XhtFOnt7s%3D&md5=f2f446e7238a0cd02bb3f1a6f277e2f6CAS |

Fu C, Liu XX, Yang WW, Zhao CM, Liu J (2016) Enhanced salt tolerance in tomato plants constitutively expressing heat-shock protein in the endoplasmic reticulum. Genetics and Molecular Research 15, gmr.15028301
Enhanced salt tolerance in tomato plants constitutively expressing heat-shock protein in the endoplasmic reticulum.CrossRef |

Guo SJ, Zhou HY, Zhang XS, Li XG, Meng QW (2007) Overexpression of CaHSP26 in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance. Journal of Plant Physiology 164, 126–136.
Overexpression of CaHSP26 in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance.CrossRef | 1:CAS:528:DC%2BD2sXjtlSrtrY%3D&md5=1cb201744645c427bb00d2671d2580f2CAS |

Halperin L, Jung J, Michalak M (2014) The many functions of the endoplasmic reticulum chaperones and folding enzymes. IUBMB Life 66, 318–326.
The many functions of the endoplasmic reticulum chaperones and folding enzymes.CrossRef | 1:CAS:528:DC%2BC2cXotV2ntLo%3D&md5=4b2d8cfa821ee2bb2c800ee6e336f91cCAS |

Havaux M, Lutz C, Grimm B (2003) Chloroplast membrane photostability in chlP transgenic tobacco plants deficient in tocopherols. Plant Physiology 132, 300–310.
Chloroplast membrane photostability in chlP transgenic tobacco plants deficient in tocopherols.CrossRef | 1:CAS:528:DC%2BD3sXktVGgsbw%3D&md5=052ac0bf1dceb6a307eef7f8128bf193CAS |

Heckathorn SA, Ryan S, Baylis JA, Wang DF, Hamilton EW, Cundiff L, Luthe DS (2002) In vivo evidence from an Agrostis stolonifera selection genotype that chloroplast small heat-shock proteins can pretoct photosystem II during heat stress. Functional Plant Biology 29, 933–944.
In vivo evidence from an Agrostis stolonifera selection genotype that chloroplast small heat-shock proteins can pretoct photosystem II during heat stress.CrossRef | 1:CAS:528:DC%2BD38XntFyqtL8%3D&md5=f41987aeb3cfd42e78bb41ebddd89640CAS |

Hermans C, Smeyers M, Rodriguez RM, Eyletters M, Strasser RJ, Delhaye JP (2003) Quality assessment of urban trees: a comparative study of physiological characterization, airborne imaging and on site fluorescence monitoring by the OJIP-test. Journal of Plant Physiology 160, 81–90.
Quality assessment of urban trees: a comparative study of physiological characterization, airborne imaging and on site fluorescence monitoring by the OJIP-test.CrossRef | 1:CAS:528:DC%2BD3sXit1SqtbY%3D&md5=0c7dccb105d232bd98f96451dace6d26CAS |

Hodges DM, Andrews CJ, Johnson DA, Hamilton RI (1997) Antioxidant enzyme and compound responses to chilling stress and their combining abilities in differentially sensitive maize hybrids. Crop Science 37, 857–863.
Antioxidant enzyme and compound responses to chilling stress and their combining abilities in differentially sensitive maize hybrids.CrossRef | 1:CAS:528:DyaK2sXkt1Olu7c%3D&md5=28b7bc968622acd474d8ccd92715943bCAS |

Howell SH (2013) Endoplasmic reticulum stress responses in plants. Annual Review of Plant Biology 64, 477–499.
Endoplasmic reticulum stress responses in plants.CrossRef | 1:CAS:528:DC%2BC3sXosFSktbs%3D&md5=c53c1b29aa4f83ebbe63c9b1b28b23a4CAS |

Jiang C, Xu J, Zhang H, Zhang X, Shi J, Li M, Ming F (2009) A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana. Plant, Cell & Environment 32, 1046–1059.
A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana.CrossRef | 1:CAS:528:DC%2BD1MXhtValtbfP&md5=435b6a93d95363259193f12cd624f344CAS |

Kamauchi S, Nakatani H, Nakano C, Urade R (2005) Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana. The FEBS Journal 272, 3461–3476.
Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana.CrossRef | 1:CAS:528:DC%2BD2MXmtFKrurY%3D&md5=200860288d7668065bbc7ef8528a6d7eCAS |

Kim DH, Xu ZY, Na YJ, Yoo YJ, Lee J, Sohn EJ, Hwang I (2011) Small heat shock protein Hsp17.8 functions as an AKR2A cofactor in the targeting of chloroplast outer membrane proteins in Arabidopsis. Plant Physiology 157, 132–146.
Small heat shock protein Hsp17.8 functions as an AKR2A cofactor in the targeting of chloroplast outer membrane proteins in Arabidopsis.CrossRef | 1:CAS:528:DC%2BC3MXht1Sit7%2FN&md5=b81ceadffb5624e4705afac90e884eefCAS |

Koizumi N, Martinez IM, Kimata Y, Kohno K, Sano H, Chrispeels MJ (2001) Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases. Plant Physiology 127, 949–962.
Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases.CrossRef | 1:CAS:528:DC%2BD3MXos1Kmsrw%3D&md5=614a9300362514053fbf5606a85a7846CAS |

Lambert W, Koeck PJ, Ahrman E, Purhonen P, Cheng K, Elmlund D, Hebert H, Emanuelsson C (2011) Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21. Protein Science 20, 291–301.
Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21.CrossRef | 1:CAS:528:DC%2BC3MXjvFWntb8%3D&md5=e24a8e535c17ae1652ab4a1123ce05e7CAS |

Li MF, Ji LS, Yang XH, Meng QW, Guo SJ (2012) The protective mechanisms of CaHSP26 in transgenic tobacco to alleviate photoinhibition of PSII during chilling stress. Plant Cell Reports 31, 1969–1979.
The protective mechanisms of CaHSP26 in transgenic tobacco to alleviate photoinhibition of PSII during chilling stress.CrossRef | 1:CAS:528:DC%2BC38XhsFSls73I&md5=bb1a2cc3841338f06824960a7ef2502dCAS |

Li H, Liu JG, Zhang LT, Pang T (2016) Antioxidant responses and photosynthetic behaviors of Kappaphycus alvarezii and Kappaphycus striatum (Rhodophyta, Solieriaceae) during low temperature stress. Botanical Studies (Taipei, Taiwan) 57, 21
Antioxidant responses and photosynthetic behaviors of Kappaphycus alvarezii and Kappaphycus striatum (Rhodophyta, Solieriaceae) during low temperature stress.CrossRef |

Liu L, Chen JY, Yang B, Wang YH (2015) Oligomer-dependent and -independent chaperone activity of sHsps in different stressed conditions. FEBS Open Bio 5, 155–162.
Oligomer-dependent and -independent chaperone activity of sHsps in different stressed conditions.CrossRef | 1:CAS:528:DC%2BC2MXktFOrs7g%3D&md5=aba62978355880a647ba2837e990a773CAS |

Ma QQ, Wang W, Li YH, Li DQ, Zou Q (2006) Alleviation of photoinhibition in drought-stressed wheat (Triticum aestivum) by foliar-applied glycinebetaine. Journal of Plant Physiology 163, 165–175.
Alleviation of photoinhibition in drought-stressed wheat (Triticum aestivum) by foliar-applied glycinebetaine.CrossRef | 1:CAS:528:DC%2BD28XhsFahtL0%3D&md5=2936bff5a7a9434ee04673dfd7df362eCAS |

Mamedov TG, Shono M (2008) Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein. Journal of Plant Research 121, 235–243.
Molecular chaperone activity of tomato (Lycopersicon esculentum) endoplasmic reticulum-located small heat shock protein.CrossRef | 1:CAS:528:DC%2BD1cXjtVGqsLk%3D&md5=9824ef0934cf2c14bf7b6b5fef700d92CAS |

Martínez IM, Chrispeels MJ (2003) Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes. The Plant Cell 15, 561–576.
Genomic analysis of the unfolded protein response in Arabidopsis shows its connection to important cellular processes.CrossRef |

McHaourab HS, Godar JA, Stewart PL (2009) Structure and mechanism of protein stability sensors: chaperone activity of small heat shock proteins. Biochemistry 48, 3828–3837.
Structure and mechanism of protein stability sensors: chaperone activity of small heat shock proteins.CrossRef | 1:CAS:528:DC%2BD1MXkt1Olsrg%3D&md5=8209017182e587fd13dd70831dea3027CAS |

Morrow G, Hightower LE, Tanguay RT (2015) Small heat shock proteins: big folding machines. Cell Stress & Chaperones 20, 207–212.
Small heat shock proteins: big folding machines.CrossRef | 1:CAS:528:DC%2BC2MXitFeqtA%3D%3D&md5=1c9574d05eb46085137250dbecacf38eCAS |

Mukherjee SP, Choudhari MA (1983) Implications of water stress induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiologia Plantarum 58, 166–170.
Implications of water stress induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings.CrossRef | 1:CAS:528:DyaL3sXksFSgt7g%3D&md5=bfd949ff3ec736be8417bd93209b85a0CAS |

Neta-Sharir I, Isaacson T, Lurie S, Weiss D (2005) Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation. The Plant Cell 17, 1829–1838.
Dual role for tomato heat shock protein 21: protecting photosystem II from oxidative stress and promoting color changes during fruit maturation.CrossRef | 1:CAS:528:DC%2BD2MXlsVynsrg%3D&md5=1311a16b85b8b559886fdd49b5fb13afCAS |

Neven LG, Haskell DW, Guy CL, Denslow N, Klein PA, Green LG, Silverman A (1992) Association of 70-kilodalton heat-shock cognate proteins with acclimation to cold. Plant Physiology 99, 1362–1369.
Association of 70-kilodalton heat-shock cognate proteins with acclimation to cold.CrossRef | 1:CAS:528:DyaK38XlvVynsLs%3D&md5=271e44da1ee8792d7b8b261f8977edbaCAS |

Ozgur R, Turkan I, Uzilday B, Sekmen AH (2014) Endoplasmic reticulum stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of Arabidopsis thaliana. Journal of Experimental Botany 65, 1377–1390.
Endoplasmic reticulum stress triggers ROS signalling, changes the redox state, and regulates the antioxidant defence of Arabidopsis thaliana.CrossRef | 1:CAS:528:DC%2BC2cXks12htLs%3D&md5=655577ad445fd6d52f414a20a8fc0948CAS |

Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience Letters 339, 62–66.
Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data.CrossRef | 1:CAS:528:DC%2BD3sXhs1Kks70%3D&md5=a9cd1d49f7c7a8c335c8b557d9d1712eCAS |

Schröder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutation Research 569, 29–63.
ER stress and the unfolded protein response.CrossRef |

Takahashi N, Sunohara Y, Fujiwara M, Matsumoto H (2017) Improved tolerance to transplanting injury and chilling stress in rice seedlings treated with orysastrobin. Plant Physiology and Biochemistry 113, 161–167.
Improved tolerance to transplanting injury and chilling stress in rice seedlings treated with orysastrobin.CrossRef | 1:CAS:528:DC%2BC2sXivFWhs7Y%3D&md5=4e13ccdc8e69aed335d3eae5678421d2CAS |

Tang ZC (1999) ‘Modern experiment protocols in plant physiology.’ (Science Press: Beijing) [In Chinese]

Treweek TM, Meehan S, Ecroyd H, Carver JA (2015) Small heat-shock proteins: important players in regulating cellular proteostasis. Cellular and Molecular Life Sciences 72, 429–451.
Small heat-shock proteins: important players in regulating cellular proteostasis.CrossRef | 1:CAS:528:DC%2BC2cXhvVOnt7rN&md5=949e00302b04d082f0a7fdd6b17589deCAS |

Ukaji N, Kuwabara C, Kanno Y, Mitsunori S, Takezawa D, Arakawa K, Fujikawa S (2010) Endoplasmic reticulum-localized small heat shock protein that accumulates in mulberry tree (Morus bombycis Koidz) during seasonal cold acclimation is responsive to abscisic acid. Tree Physiology 30, 502–513.
Endoplasmic reticulum-localized small heat shock protein that accumulates in mulberry tree (Morus bombycis Koidz) during seasonal cold acclimation is responsive to abscisic acid.CrossRef | 1:CAS:528:DC%2BC3cXmtVCisLg%3D&md5=9147371013bf6d114620e84a86bffe15CAS |

ur Rahman H, Malik SA, Saleem M (2004) Heat tolerance of upland cotton during fruiting stage evaluated using cellular membrane thermostability. Field Crops Research 85, 149–158.
Heat tolerance of upland cotton during fruiting stage evaluated using cellular membrane thermostability.CrossRef |

Valente MA, Faria JA, Soares-Ramos JR, Reis PA (2009) The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco. Journal of Experimental Botany 60, 533–546.
The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco.CrossRef | 1:CAS:528:DC%2BD1MXivFSmtLY%3D&md5=e12e699e8edb6f3688f5c5b34437e06bCAS |

Wan S, Jiang L (2016) Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in plants. Protoplasma 253, 753–764.
Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in plants.CrossRef | 1:CAS:528:DC%2BC2MXhtVKnu7zM&md5=a2954b7154266408098debfb358e0348CAS |

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 | 1:CAS:528:DC%2BD2cXjvVemtbw%3D&md5=9202a77f8308dcc00d3fa04b97151131CAS |

Waters ER (2013) The evolution, function, structure, and expression of the plant sHSPs. Journal of Experimental Botany 64, 391–403.
The evolution, function, structure, and expression of the plant sHSPs.CrossRef | 1:CAS:528:DC%2BC3sXntVGksw%3D%3D&md5=b53d00fa27d3dc74d177963b373faf71CAS |

Xu JH, Zhang M, Liu G, Yang XP, Hou XL (2016) Comparative transcriptome profiling of chilling stress responsiveness in grafted watermelon seedlings. Plant Physiology and Biochemistry 109, 561–570.
Comparative transcriptome profiling of chilling stress responsiveness in grafted watermelon seedlings.CrossRef | 1:CAS:528:DC%2BC28XhvVarsb3L&md5=273e612b4c59bc92e5f6260788268d1aCAS |

Xue M, Guo YY, Yan Q, Qin D, Lu C (2013) Preparation and application of polyclonal antibodies against KSH V v–cyclin. Journal of Biomedical Research 27, 421–429.

Yang JS, Wang R, Meng JJ, Bi YP, Xu PL, Guo F, Wan SB, He QW, Li XG (2010) Overexpression of Arabidopsis CBF1 gene in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance. Journal of Plant Physiology 167, 534–539.
Overexpression of Arabidopsis CBF1 gene in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance.CrossRef | 1:CAS:528:DC%2BC3cXlt1Krsrs%3D&md5=d1737e6ea55a11dc909555c86e814d9dCAS |

Zhao CM, Shono M, Sun AQ, Yi SY, Li MH, Liu J (2007) Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato. Journal of Plant Physiology 164, 835–841.
Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato.CrossRef | 1:CAS:528:DC%2BD2sXoslSisbY%3D&md5=481420a5cafad0721b946090fa4da01bCAS |

Zhao HL, Ye L, Wang YP, Zhou XT, Yang JW, Wang JW, Cao K, Zou ZR (2016) Melatonin increases the chilling tolerance of chloroplast in cucumber seedlings by regulating photosynthetic electron flux and the ascorbate–glutathione cycle. Frontiers in Plant Science 7, 1814
Melatonin increases the chilling tolerance of chloroplast in cucumber seedlings by regulating photosynthetic electron flux and the ascorbate–glutathione cycle.CrossRef |

Zong XJ, Li DP, Gu LK, Liu LX, Hu XL, Li DQ (2009) Abscisic acid and hydrogen peroxide induce a novel maize group C MAP kinase gene, ZmMPK7, which is responsible for the removal of reactive oxygen species. Planta 229, 485–495.
Abscisic acid and hydrogen peroxide induce a novel maize group C MAP kinase gene, ZmMPK7, which is responsible for the removal of reactive oxygen species.CrossRef | 1:CAS:528:DC%2BD1MXhtFSmsrk%3D&md5=b48b1a31a913796155469ab01b2bfe90CAS |



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