Cell Stress and Chaperones

, Volume 19, Issue 6, pp 939–948 | Cite as

Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause

  • Jantina Toxopeus
  • Alden H. Warner
  • Thomas H. MacRaeEmail author
Original Paper


Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis—life without water—during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H2O2) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.


Late embryogenesis abundant (LEA) proteins Intrinsically disordered proteins (IDPs) Desiccation tolerance Freeze tolerance Brine shrimp RNA interference (RNAi) 



This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN/7661-2011) to THM, and an NSERC Julie Payette Scholarship and Killam Pre-doctoral Scholarship awarded to JT.


  1. Amara I, Capellades M, Ludevid MD, Pagès M, Goday A (2013) Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene. J Plant Physiol 170:864–873PubMedCrossRefGoogle Scholar
  2. National Oceanic and Atmospheric Administration (2011). NOAA’s 1981-2010 Climate Normals. Accessed 1 August 2013
  3. Bagshaw JC, Rafiee P, Matthews CO, MacRae TH (1986) Cadmium and zinc reversibly arrest development of Artemia larvae. Bull Environ Contam Toxicol 37:289–296PubMedCrossRefGoogle Scholar
  4. Bahrndorff S, Tunnacliffe A, Wise MJ, McGee B, Holmstrup M, Loeshcke V (2009) Bioinformatics and protein expression analyses implicates LEA proteins in the drought responses of Collembola. J Insect Physiol 55:210–217PubMedCrossRefGoogle Scholar
  5. Battista JR, Park M-J, McLemore AE (2001) Inactivation of two homologues of proteins presumed to be involved in the desiccation tolerance of plants sensitizes Deinococcus radiodurans R1 to desiccation. Cryobiology 43:133–139PubMedCrossRefGoogle Scholar
  6. Boswell LC, Moore DS, Hand SC (2013) Quantification of cellular protein expression and molecular features of group 3 LEA proteins from embryos of Artemia franciscana. Cell Stress Chaperones. doi: 10.1007/s12192-013-0458-3 PubMedCentralPubMedGoogle Scholar
  7. Browne J, Tunnacliffe A, Burnell A (2002) Anhydrobiosis: plant desiccation gene found in a nematode. Nature 416:38PubMedCrossRefGoogle Scholar
  8. Buitink J, Leprince O (2004) Glass formation in plant anhydrobiotes: survival in the dry state. Cryobiology 48:215–228PubMedCrossRefGoogle Scholar
  9. Caprioli M, Katholm AK, Melone G, Ramløv H, Ricci C, Santo N (2004) Trehalose in desiccated rotifers: a comparison between a bdelloid and a monogonont species. Comp Biochem Physiol A 139:527–532CrossRefGoogle Scholar
  10. Chakrabortee S, Boschetti C, Walton LJ, Sarkar S, Rubinsztein DC, Tunnacliffe A (2007) Hydrophilic protein associated with desiccation tolerance exhibits board stabilization function. Proc Natl Acad Sci USA 104:19073–18078CrossRefGoogle Scholar
  11. Chakrabortee S, Tripathi R, Watson M, Kaminski Schierle GS, Kurniawan DP, Kaminski CF, Wise MJ, Tunnacliffe A (2012) Intrinsically disordered proteins as molecular shields. Mol Biosys 8:210–219CrossRefGoogle Scholar
  12. Clegg JS (1986) The physical properties and metabolic status of Artemia cysts at low water contents: the ‘water replacement hypothesis’. In: Leopold AC (ed) Membranes, metabolism and dry organisms. Comstock, Ithaca, pp 169–187Google Scholar
  13. Clegg JS, Trotman CNA (2002) Physiology and biochemistry of Artemia ecology. In: Abatzopolous TJ, Beardmore JA, Clegg JS, Sorgeloos P (eds) Artemia: basic and applied biology. Kluwer, Dordrecht, pp 129–170CrossRefGoogle Scholar
  14. Clegg JS, Drinkwater LE, Sorgeloos P (1996) The metabolic status of diapause embryos of Artemia franciscana (SFB). Physiol Zool 69:49–66Google Scholar
  15. Copf T, Schröder R, Averof M (2004) Ancestral role of caudal genes in axis elongation and segmentation. Proc Natl Acad Sci 101:17711–17715PubMedCentralPubMedCrossRefGoogle Scholar
  16. Cornette R, Kanamori Y, Watanabe M, Nakahara Y, Gusev O, Mistumasu K, Kadono-Okuda K, Shimomura M, Mita K, Kikawada T, Okuda T (2010) Identification of anhydrobiosis-related genes from an expressed sequence tag database in the cryptobiotic midge Polypedilum vanderplanki (Diptera; Chironomidae). J Biol Chem 285:35889–35899PubMedCentralPubMedCrossRefGoogle Scholar
  17. Crowe JH, Crowe LM, O’Dell SJ (1981) Ice formation during freezing of Artemia cysts of variable water contents. Mol Physiol 1:145–152Google Scholar
  18. Dai L, Chen D-F, Liu Y-L, Zhao Y, Yang F, Yang J-S, Yang W-J (2011) Extracellular matrix peptides of Artemia cyst shell participate in protecting encysted embryos from extreme environments. PLoS ONE 6(6):e20187PubMedCentralPubMedCrossRefGoogle Scholar
  19. de Chaffoy D, de Maeyer-Criel G, Kondo M (1978) On the permeability and formation of the embryonic cuticle during development in vivo and in vitro of Artemia salina embryos. Differentiation 12:99–109CrossRefGoogle Scholar
  20. Drinkwater LE, Crowe JH (1987) Regulation of embryonic diapause in Artemia: environmental and physiological signals. J Exp Zool 241:297–307CrossRefGoogle Scholar
  21. Duan J, Cai W (2012) OsLEA3-2, an abiotic stress gene of rice plays a key role in salt and drought tolerance. PLoS ONE 7(9):e45117PubMedCentralPubMedCrossRefGoogle Scholar
  22. Erkut C, Vasilj A, Boland S, Habermann B, Shevchenko A, Kurzchalia TV (2013) Molecular strategies of the Caenorhabditis elegans dauer larva to survive extreme desiccation. PLoS ONE 8(12):e82473PubMedCentralPubMedCrossRefGoogle Scholar
  23. Förster F, Liang C, Shkumatov A, Beisser D, Engelmann JC, Schnölzer M, Frohme M, Müller T, Schill RO, Dandekar T (2009) Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades. BMC Genomics 10:469PubMedCentralPubMedCrossRefGoogle Scholar
  24. Furuki T, Shimizu T, Chakrabortee S, Yamakawa K, Hatanaka R, Takahashi T, Kikawada T, Okuda T, Mihara H, Tunnacliffe A, Sakurai M (2012) Effects of Group 3 LEA model peptides on desiccation-induced protein aggregation. Biochim Biophys Acta 1824:891–897PubMedCrossRefGoogle Scholar
  25. Go EC, Pandey AS, MacRae TH (1990) Effect of inorganic mercury on the emergence and hatching of the brine shrimp Artemia franciscana. Mar Biol 107:93–102CrossRefGoogle Scholar
  26. Goyal K, Tisi L, Basran A, Browne J, Burnell A, Zurdo J, Tunnacliffe A (2003) Transition from natively unfolded to folded state induced by desiccation in an anhydrobiotic nematode protein. J Biol Chem 278:12977–12984PubMedCrossRefGoogle Scholar
  27. Goyal K, Walton LJ, Tunnacliffe A (2005) LEA proteins prevent protein aggregation due to water stress. Biochem J 388:151–157PubMedCentralPubMedCrossRefGoogle Scholar
  28. Grelet J, Benamar A, Teyssier E, Avelange-Macherel M-H, Grunwald D, Macherel D (2005) Identification in pea seed mitochondria of a late embryogenesis abundant protein able to protect enzymes from drying. Plant Physiol 137:157–167PubMedCentralPubMedCrossRefGoogle Scholar
  29. Hand SC, Jones D, Menze MA, Witt TL (2007) Life without water: expression of plant LEA genes by an anhydrobiotic arthropod. J Exp Zool 307A:62–66CrossRefGoogle Scholar
  30. Hand SC, Menze MA, Toner M, Boswell L, Moore D (2011) LEA proteins during water stress: not just for plants anymore. Annu Rev Physiol 73:115–134PubMedCrossRefGoogle Scholar
  31. Hatanaka R, Hagiwara-Komoda Y, Furuki T, Kanamori Y, Fujita M, Cornette R, Sakurai M, Okuda T, Kikawada T (2013) An abundant LEA protein in the anhydrobiotic midge, PvLEA4, acts as a molecular shield by limiting growth of aggregating protein particles. Insect Biochem Mol Biol 43:1055–1067PubMedCrossRefGoogle Scholar
  32. Hengherr S, Schill RO, Clegg JS (2011) Mechanisms associated with cellular desiccation tolerance in the animal extremophile Artemia. Physiol Biochem Zool 84:249–257PubMedCrossRefGoogle Scholar
  33. Hincha DK, Thalhammer A (2012) LEA proteins: IDPs with versatile functions in cellular dehydration tolerance. Biochem Soc Tran 40:1000–1003CrossRefGoogle Scholar
  34. Hundertmark M, Popova AV, Rausch S, Seckler R, Hincha DK (2012) Influence of drying on the secondary structure of intrinsically disordered and globular proteins. Biochem Biophys Res Commun 417:122–128PubMedCrossRefGoogle Scholar
  35. King AM, MacRae TH (2012) The small heat shock protein p26 aids development of encysting Artemia embryos, prevents spontaneous diapause termination and aids in stress. PLoS ONE 7(8):e43723PubMedCentralPubMedCrossRefGoogle Scholar
  36. King AM, Toxopeus J, MacRae TH (2013) Functional differentiation of small heat shock proteins in diapause-destined Artemia embryos. FEBS J 280:4761–4772PubMedCrossRefGoogle Scholar
  37. King AM, Toxopeus J, MacRae TH (2014) Artemin, a diapause-specific chaperone, contributes to stress tolerance of Artemia cysts and influences their release from females. J Exp Biol. doi: 10.1242/eb100081 Google Scholar
  38. Lapinski J, Tunncliffe A (2003) Anhydrobiosis without trehalose in bdelloid rotifers. FEBS Lett 553:387–390PubMedCrossRefGoogle Scholar
  39. Li S, Chakraborty N, Borcar A, Menze MA, Toner M, Hand S (2012) Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation. Proc Natl Acad Sci USA 100:20859–20864CrossRefGoogle Scholar
  40. Liang P, MacRae TH (1999) The synthesis of small heat shock/α-crystallin protein in Artemia and its relationship to stress tolerance during development. Dev Biol 207:445–456PubMedCrossRefGoogle Scholar
  41. Loi P, Iuso D, Czernik M, Zacchini F, Ptak G (2013) Towards storage of cells and gametes in dry form. Trends Biotech 31:688–695CrossRefGoogle Scholar
  42. Ma W-M, Li H-W, Dai Z-M, Yang J-S, Yang F, Yang W-J (2013) Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells. Biochem J 449:285–294PubMedCrossRefGoogle Scholar
  43. Manfre AJ, Lanni LM, Marcotte WR (2006) The Arabidopsis group 1 LATE EMBRYOGENESIS ABUNDANT protein ATEM6 is required for normal seed development. Plant Physiol 140:140–149PubMedCentralPubMedCrossRefGoogle Scholar
  44. Marunde MR, Samarajeewa DA, Anderson J, Li S, Hand SC, Menze MA (2013) Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein. J Insect Physiol 59:377–386PubMedCrossRefGoogle Scholar
  45. Menze MA, Boswell L, Toner M, Hand SC (2009) Occurrence of mitochondria-targeted late embryogenesis abundant (LEA) gene in animals increases organelle resistance to water stress. J Biol Chem 284:10714–10719PubMedCentralPubMedCrossRefGoogle Scholar
  46. Mowla SB, Cuypers A, Driscoll SP, Kiddle G, Thomson J, Foyer CH, Theodoulou FL (2006) Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)-like protein in oxidative stress tolerance. Plant J 48:743–756PubMedCrossRefGoogle Scholar
  47. Pandey AS, MacRae TH (1991) Toxicity of organic mercury compounds to the developing brine shrimp, Artemia. Ecotoxic Environ Saf 21:68–79CrossRefGoogle Scholar
  48. Qiu Z, MacRae TH (2008) ArHsp22, a developmentally regulated small heat shock protein produced in diapause-destined Artemia embryos, is stress inducible in adults. FEBS J 275:3556–3566PubMedCrossRefGoogle Scholar
  49. Qiu Z, MacRae TH (2010) A molecular overview of diapause in embryos of the crustacean, Artemia franciscana. In: Clark M, Cerda J, Lubzens E (eds) Dormancy and resistance in harsh environments. Springer, Berlin, pp 165–187CrossRefGoogle Scholar
  50. Rafiee P, Matthews CO, Bagshaw JC, MacRae TH (1986) Reversible arrest of Artemia development by cadmium. Can J Zool 64:1633–1641CrossRefGoogle Scholar
  51. Ramløv H, Hvidt A (1992) Artemia cysts at subzero temperatures studied by differential scanning calorimetry. Cryobiology 29:131–137CrossRefGoogle Scholar
  52. Reyes JL, Rodrigo M-J, Colmenero-Flores JM, Gil J-V, Garay-Arroyo A, Campos F, Salamini F, Bartels D, Covarrubias AA (2005) Hydrophilins from distant organisms can protect enzymatic activities from water limitation effects in vitro. Plant Cell Environ 28:709–718CrossRefGoogle Scholar
  53. Robbins HM, Van Stappen G, Sorgeloos P, Sung YY, MacRae TH, Bossier P (2010) Diapause termination and development of encysted Artemia embryos: roles for nitric oxide and hydrogen peroxide. J Exp Biol 213:1464–1470PubMedCrossRefGoogle Scholar
  54. Sakurai M, Furuki T, Akao K, Tanaka D, Nakahara Y, Kikawada T, Watanabe M, Okuda T (2008) Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki. Proc Natl Acad Sci USA 105:5093–5098PubMedCentralPubMedCrossRefGoogle Scholar
  55. Sharon MA, Kozarova A, Clegg JS, Vacratsis PO, Warner AH (2009) Characterization of a group 1 late embryogenesis abundant protein in encysted embryos of the brine shrimp Artemia franciscana. Biochem Cell Biol 87:415–430PubMedCrossRefGoogle Scholar
  56. Shimizu T, Kanamori Y, Furuki T, Kikawada T, Okuda T, Takahashi T, Mihara H, Sakurai M (2010) Desiccation-induced structuralization and glass formation of group 3 late embryogenesis abundant protein model peptides. Biochemistry 49:1093–1104PubMedCrossRefGoogle Scholar
  57. Soulages JL, Kim K, Walters C, Cushman JC (2002) Temperature-induced extended helix/random coil transitions in a group 1 late embryogenesis-abundant protein from soybean. Plant Physiol 128:822–832PubMedCentralPubMedCrossRefGoogle Scholar
  58. Storey KB, Storey JM (2013) Molecular biology of freezing tolerance. Comp Physiol 3:1283–1308Google Scholar
  59. Tolleter D, Hincha DK, Macherel D (2010) A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state. Biochim Biophys Acta 1798:1926–1933PubMedCrossRefGoogle Scholar
  60. Tompa K, Bokor M, Tompa P (2010) Hydration of intrinsically disordered proteins from wide-line NMR. In: Uversky V, Longhi S (eds) Instrumental analysis of intrinsically disordered proteins: assessing structure and conformation. Wiley, Hoboken, pp 345–368CrossRefGoogle Scholar
  61. Trotman CNA, Mansfield BC, Tate WP (1980) Inhibition of emergence, hatching, and protein biosynthesis in embryonic Artemia salina. Dev Biol 80:167–174PubMedCrossRefGoogle Scholar
  62. Tunnacliffe A, Wise MJ (2007) The continuing conundrum of the LEA proteins. Naturwissenschaften 94:791–812PubMedCrossRefGoogle Scholar
  63. Tunnacliffe A, Lapinski J, McGee B (2005) A putative LEA protein, but no trehalose, is present in anhydrobiotic bdelloid rotifers. Hydrobiologia 546:315–321CrossRefGoogle Scholar
  64. Tyson T, O’Mahony Zamora G, Wong S, Skelton M, Daly B, Jones JT, Mulvihill ED, Elsworth B, Phillips M, Blaxter M, Burnel AM (2012) A molecular analysis in the anhydrobiotic nematode Panagrolaimus superbus using expressed sequence tags. BMC Res Notes 5:68–91PubMedCentralPubMedCrossRefGoogle Scholar
  65. Van Stappen G, Lavens P, Sorgeloos P (1998) Effects of hydrogen peroxide treatment in Artemia cysts of different geographical origin. Arch Hydrobiol Spec Issues Advanc Limnol 52:281–296Google Scholar
  66. Veeramani S, Baskaralingam V (2011) Shell-bound iron dependant nitric oxide synthesis in encysted Artemia parthenogenetica embryos during hydrogen peroxide exposure. Biometals 24:1035–1044PubMedCrossRefGoogle Scholar
  67. Warner AH, Miroshnychenko O, Kozarova A, Vacratsis PO, MacRae TH, Kim J, Clegg JS (2010) Evidence for multiple group 1 late embryogenesis abundant proteins in encysted embryos of Artemia and their organelles. J Biochem 148:581–592PubMedCrossRefGoogle Scholar
  68. Warner AH, Chakrabortee S, Tunnacliffe A, Clegg JS (2012) Complexity of the heat-soluble LEA proteome in Artemia species. Comp Biochem Physiol D 7:260–267Google Scholar
  69. Wolkers WF, McCready S, Brandt WF, Lindsey GG, Hoekstra FA (2001) Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro. Biochim Biophys Acta 1544:196–206PubMedCrossRefGoogle Scholar
  70. Wu G, Zhang H, Sun J, Liu F, Ge X, Chen W-H, Yu J, Wang W (2011) Diverse LEA (late embryogenesis abundant) and LEA-like genes and their responses to hypersaline stress in post-diapause embryonic development of Artemia franciscana. Comp Biochem Physiol B 160:32--39Google Scholar
  71. Yoshida T, Arii Y, Hino K, Sawatani I, Tanaka M, Takahashi R, Bando T, Mukai K, Fukuo K (2011) High hatching rates after cryopreservation of hydrated cysts of the brine shrimp A. franciscana. Cryoletters 32:206–215PubMedGoogle Scholar
  72. Zhao Y, Ding X, Xiang Y, Dai Z-M, Yang J-S, Yang W-J (2012) Involvement of cyclin K posttranscriptional regulation in the formation of Artemia diapause cysts. PLoS ONE 7(2):e32129PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Cell Stress Society International 2014

Authors and Affiliations

  • Jantina Toxopeus
    • 1
  • Alden H. Warner
    • 2
  • Thomas H. MacRae
    • 1
    Email author
  1. 1.Department of BiologyDalhousie UniversityHalifaxCanada
  2. 2.Department of Biological SciencesUniversity of WindsorWindsorCanada

Personalised recommendations