Insights into Maize LEA proteins: from proteomics to functional approaches.

LEA (late embryogenesis abundant) proteins participate in plant stress tolerance responses, but the mechanisms by which protection occurs are not fully understood. In the present work the unfolded proteins from maize dry embryos were analyzed by mass spectrometry. Twenty embryo proteins were identified, and among them 13 corresponded to LEA-type proteins. We selected three major LEA proteins, Emb564, Rab17 and Mlg3, belonging to groups 1, 2 and 3, respectively, and we undertook a comparative study in order to highlight differences among them. The post-translational modifications of native proteins were analyzed and the anti-aggregation properties of recombinant Emb564, Rab17 and Mgl3 proteins were evaluated in vitro. In addition, the protective effects of the LEA proteins were assessed in living cells under stress in Escherichia coli cells and in Nicotiana bentamiana leaves agroinfiltrated with fluorescent LEA-green fluorescent protein (GFP) fusions. Protein visualization by confocal microscopy indicated that cells expressing Mg3-GFP showed reduced cell shrinkage effects during dehydration and that Rab17-GFP co-localized to leaf oil bodies after heat shock. Overall, the results highlight differences and suggest functional diversity among maize LEA groups.

[1]  S. Fowler,et al.  Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O. , 1985, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[2]  R. Eritja,et al.  Phosphorylation of maize RAB-17 protein by casein kinase 2. , 1991, The Journal of biological chemistry.

[3]  T. Grudt,et al.  Abscisic Acid and the developmental regulation of embryo storage proteins in maize. , 1991, Plant physiology.

[4]  J. Tzen,et al.  Characterization of the charged components and their topology on the surface of plant seed oil bodies. , 1992, The Journal of biological chemistry.

[5]  M. Mar Albà,et al.  The maize abscisic acid-responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals. , 1994, The Plant cell.

[6]  M. Mar Albà,et al.  Expression and cellular localization of rab28 mRNA and Rab28 protein during maize embryogenesis. , 1996, The Plant journal : for cell and molecular biology.

[7]  E. Chen,et al.  A new method for seed oil body purification and examination of oil body integrity following germination. , 1997, Journal of biochemistry.

[8]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[9]  Jean Danyluk,et al.  Accumulation of an Acidic Dehydrin in the Vicinity of the Plasma Membrane during Cold Acclimation of Wheat , 1998, Plant Cell.

[10]  F. Salamini,et al.  Desiccation tolerance in the resurrection plant Craterostigma plantagineum. A contribution to the study of drought tolerance at the molecular level. , 2001, Plant physiology.

[11]  Elizabeth A. Smith,et al.  The Calcium-Binding Activity of a Vacuole-Associated, Dehydrin-Like Protein Is Regulated by Phosphorylation1 , 2002, Plant Physiology.

[12]  M. Alsheikh,et al.  Ion Binding Properties of the Dehydrin ERD14 Are Dependent upon Phosphorylation* , 2003, Journal of Biological Chemistry.

[13]  T. Close,et al.  The binding of Maize DHN1 to Lipid Vesicles. Gain of Structure and Lipid Specificity1 , 2003, Plant Physiology.

[14]  Michael J. Wise,et al.  LEAping to conclusions: A computational reanalysis of late embryogenesis abundant proteins and their possible roles , 2003, BMC Bioinformatics.

[15]  M. Pagés,et al.  Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Ruthie Angelovici,et al.  Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. , 2004, The Plant journal : for cell and molecular biology.

[17]  N. Mock,et al.  An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue , 1994, Plant Cell, Tissue and Organ Culture.

[18]  Yinsheng Wang,et al.  Beta-elimination coupled with tandem mass spectrometry for the identification of in vivo and in vitro phosphorylation sites in maize dehydrin DHN1 protein. , 2004, Biochemistry.

[19]  M. Wise,et al.  POPP the question: what do LEA proteins do? , 2004, Trends in plant science.

[20]  P. Mäkelä,et al.  Overexpression of Multiple Dehydrin Genes Enhances Tolerance to Freezing Stress in Arabidopsis , 2004, Plant Molecular Biology.

[21]  R. Savé,et al.  Maize Rabl7 overexpression in Arabidopsis plants promotes osmotic stress tolerance , 2004 .

[22]  Marc S. Cortese,et al.  Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment. , 2005, Journal of proteome research.

[23]  T. Close,et al.  Nuclear and cytoplasmic localization of maize embryo and aleurone dehydrin , 1994, Protoplasma.

[24]  D. Cai,et al.  Expression in Escherichia coli of Three Different Soybean Late Embryogenesis Abundant (LEA) Genes to Investigate Enhanced Stress Tolerance , 2005 .

[25]  F. Salamini,et al.  Hydrophilins from distant organisms can protect enzymatic activities from water limitation effects in vitro , 2005 .

[26]  Hélène Rogniaux,et al.  Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance1[W] , 2006, Plant Physiology.

[27]  Rodrigo M. P. Siloto,et al.  The Accumulation of Oleosins Determines the Size of Seed Oilbodies in Arabidopsis[W][OA] , 2006, The Plant Cell Online.

[28]  M. Pagés,et al.  Towards the identification of late‐embryogenic‐abundant phosphoproteome in Arabidopsis by 2‐DE and MS , 2006, Proteomics.

[29]  J. D. Curtis,et al.  Oil bodies in leaf mesophyll cells of angiosperms: overview and a selected survey. , 2006, American journal of botany.

[30]  Vishwajeeth R Pagala,et al.  Proteomic studies of the intrinsically unstructured mammalian proteome. , 2006, Journal of proteome research.

[31]  D. Bartels,et al.  Desiccation of the resurrection plant Craterostigma plantagineum induces dynamic changes in protein phosphorylation. , 2006, Plant, cell & environment.

[32]  A. Covarrubias,et al.  Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth. , 2006, Biochemical and biophysical research communications.

[33]  M. A. Odena,et al.  LC-MSMS identification of Arabidopsis thaliana heat-stable seed proteins: enriching for LEA-type proteins by acid treatment. , 2007, Journal of mass spectrometry : JMS.

[34]  M. Wise,et al.  The continuing conundrum of the LEA proteins , 2007, Naturwissenschaften.

[35]  D. Hincha,et al.  LEA (Late Embryogenesis Abundant) proteins and their encoding genes in Arabidopsis thaliana , 2008, BMC Genomics.

[36]  S. Delrot,et al.  An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells. , 2007, Biochimica et biophysica acta.

[37]  S. Yuasa,et al.  Intramolecular Control of Protein Stability, Subnuclear Compartmentalization, and Coactivator Function of Peroxisome Proliferator-activated Receptor γ Coactivator 1α* , 2007, Journal of Biological Chemistry.

[38]  Kelly M. Hines,et al.  A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying. , 2007, Plant physiology and biochemistry : PPB.

[39]  M. Jaquinod,et al.  Structure and Function of a Mitochondrial Late Embryogenesis Abundant Protein Are Revealed by Desiccation[W] , 2007, The Plant Cell Online.

[40]  D. Rubinsztein,et al.  Hydrophilic protein associated with desiccation tolerance exhibits broad protein stabilization function , 2007, Proceedings of the National Academy of Sciences.

[41]  J. Mouillon,et al.  Mimicking the Plant Cell Interior under Water Stress by Macromolecular Crowding: Disordered Dehydrin Proteins Are Highly Resistant to Structural Collapse1[W] , 2008, Plant Physiology.

[42]  A. Covarrubias,et al.  The Enigmatic LEA Proteins and Other Hydrophilins1[W] , 2008, Plant Physiology.

[43]  M. Delseny,et al.  Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana , 2008, Plant Molecular Biology.

[44]  Y. Hsing,et al.  Late Embryogenesis Abundant Proteins , 2008 .

[45]  P. Tompa,et al.  Disordered plant LEA proteins as molecular chaperones , 2008, Plant signaling & behavior.

[46]  Hui Wei,et al.  Functional dissection of hydrophilins during in vitro freeze protection. , 2008, Plant, cell & environment.

[47]  T. Close,et al.  The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes1[W][OA] , 2009, Plant Physiology.

[48]  Xiaoming He,et al.  Desiccation induced structural alterations in a 66-amino acid fragment of an anhydrobiotic nematode late embryogenesis abundant (LEA) protein. , 2009, Biomacromolecules.

[49]  Chien-Yu Huang,et al.  Oil Bodies and Oleosins in Physcomitrella Possess Characteristics Representative of Early Trends in Evolution1[W][OA] , 2009, Plant Physiology.

[50]  W. Marcotte,et al.  Seed dehydration and the establishment of desiccation tolerance during seed maturation is altered in the Arabidopsis thaliana mutant atem6-1. , 2008, Plant & cell physiology.

[51]  V. Chinnusamy,et al.  Abiotic stress and ABA-inducible Group 4 LEA from Brassica napus plays a key role in salt and drought tolerance. , 2009, Journal of biotechnology.

[52]  I. Hara-Nishimura,et al.  Oil-body-membrane proteins and their physiological functions in plants. , 2010, Biological & pharmaceutical bulletin.

[53]  J. de Dios Alché,et al.  Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination , 2010, Journal of experimental botany.

[54]  D. Hincha,et al.  LEA Proteins: Versatility of Form and Function , 2010 .

[55]  A. Warner,et al.  Evidence for multiple group 1 late embryogenesis abundant proteins in encysted embryos of Artemia and their organelles. , 2010, Journal of biochemistry.

[56]  M. Clark,et al.  Dormancy and Resistance in Harsh Environments , 2010 .

[57]  J. Boudet,et al.  MtPM25 is an atypical hydrophobic late embryogenesis-abundant protein that dissociates cold and desiccation-aggregated proteins. , 2010, Plant, cell & environment.

[58]  Peter Tompa,et al.  Intrinsically disordered chaperones in plants and animals. , 2010, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[59]  Alain Vavasseur,et al.  RD20, a stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana. , 2010, Plant & cell physiology.

[60]  M. Sakurai,et al.  Desiccation-induced structuralization and glass formation of group 3 late embryogenesis abundant protein model peptides. , 2010, Biochemistry.

[61]  K. Gupta,et al.  SbDREB2A, an A-2 type DREB transcription factor from extreme halophyte Salicornia brachiata confers abiotic stress tolerance in Escherichia coli , 2010, Plant Cell Reports.

[62]  M. Toner,et al.  LEA proteins during water stress: not just for plants anymore. , 2011, Annual review of physiology.

[63]  G. Harauz,et al.  Phosphorylation of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 facilitates cation-induced conformational changes and actin assembly. , 2011, Biochemistry.

[64]  G. Gröbner,et al.  Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein[W] , 2011, Plant Cell.