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MS-desi, a desiccation-related protein in the floral nectar of the evergreen velvet bean (Mucuna sempervirens Hemsl): molecular identification and characterization

Abstract

Plant desiccation-related proteins (DRPs) were first identified as pcC13-62 from the resurrection plant Craterostigma plantagineum and it has been suggested they are involved in plant desiccation tolerance. We identified and characterized a plant DRP, which we called MS-desi, in the floral nectar of a subtropical bean species, Mucuna sempervirens (MS). MS-desi is a major nectar protein (nectarin) of the bean plant and expresses exclusively in the stylopodium, where the nectary is located. The full-length MS-desi gene encodes for a protein of 306 amino acids with a molecular mass of 33,248 Da, and possesses a ferritin-like domain and a signal peptide of 30 amino acids. Structural and phylogenetic analysis demonstrated MS-desi has high similarity to members of the plant DRPs, including pcC 13-62 protein. MS-desi has a similar hydropathy profile to that of pcC13-62 with a grand average of hydropathy index of 0.130 for MS-desi and 0.106 for pcC13-62 protein, which is very different from those of dehydrins and late embryogenesis abundant proteins. The protein’s secondary structures, both predicted from the amino acid sequence and directly analysed by far UV circular dichroism, showed that MS-desi is mainly composed of alpha helices and is relatively temperature dependent. The structure change is reversible within a wide range of temperatures. Purified MS-desi and raw MS floral nectar showed dose-dependent citrate synthase inhibition activity, but insensitivity to lactate dehydrogenase, suggesting that, unlike dehydrins, it does not act as a chaperone. The overall results constitute, to our knowledge, the first study on a desiccation-related protein in plant floral nectar.

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Abbreviations

ABA:

Abscisic acid

CBB:

Coomassie brilliant blue

CD:

Circular dichroism

CS:

Citrate synthase

DRPs:

Plant desiccation-related proteins

DTT:

Dithiothreitol

GRAVY:

Grand average of hydropathy

IPG:

Immobilized pH gradient

LDH:

Lactate dehydrogenase

LEA:

Late embryogenesis abundant

MALDI-TOF:

Matrix-assisted laser desorption ionization-time of flight

NJ:

Neighbor-joining

PVDF:

Polyvinylidene difluoride

References

  1. Allen ON, Allen EK (1981) The Leguminosae: a source book of characteristics, uses, and nodulation. University of Wisconsin Press, Madison

  2. Bae EK, Lee H, Lee JS, Noh EW (2010) Isolation and characterization of osmotic stress-induced genes in poplar cells by suppression subtractive hybridization and cDNA microarray analysis. Plant Physiol Biochem 48:136–141

  3. Bardel J, Louwagie M, Jaquinod M, Jourdain A, Luche S, Rabilloud T, Macherel D, Garin J, Bourguignon J (2002) A survey of the plant mitochondrial proteome in relation to development. Proteomics 2:880–898

  4. Battaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias AA (2008) The enigmatic LEA proteins and other hydrophilins. Plant Physiol 148:6–24

  5. Battista JR, Park MJ, 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 43:133–139

  6. Bernardello G (2007) A systematic survey of floral nectaries. In: Nicolson SW, Nepi M, Pacini E (eds) Nectaries and nectar. Springer, Amsterdam, pp 19–128

  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  8. Carter C, Thornburg RW (2004) Is the nectar redox cycle a floral defense against microbial attack? Trends Plant Sci 9:320–324

  9. Carter C, Graham RA, Thornburg RW (1999) Nectarin I is a novel, soluble germin-like protein expressed in the nectar of Nicotiana sp. Plant Mol Biol 41:207–216

  10. Castellanos-Serra L, Proenza W, Huerta V, Moritz RL, Simpson RJ (1999) Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts. Electrophoresis 20:732–737

  11. Cole C, Barber JD, Barton GJ (2008) The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36:W197–W201

  12. Collett H, Shen A, Gardner M, Farrant JM, Denby KJ, Illing N (2004) Towards transcript profiling of desiccation tolerance in Xerophyta humilis: construction of a normalized 11 k X. humilis cDNA set and microarray expression analysis of 424 cDNAs in response to dehydration. Physiol Plant 122:39–53

  13. Combet C, Blanchet C, Geourjon C, Deleage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150

  14. Dai X, Wang G, Yang DS et al (2010) TrichOME: a comparative omics database for plant trichomes. Plant Physiol 152:44–54

  15. Dubois M, Gilles KA, Hamilton JR, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

  16. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971

  17. Gonzalez-Teuber M, Eilmus S, Muck A, Svatos A, Heil M (2009) Pathogenesis-related proteins protect extrafloral nectar from microbial infestation. Plant J 58:464–473

  18. Goyal K, Walton LJ, Tunnacliffe A (2005) LEA proteins prevent protein aggregation due to water stress. Biochem J 388:151–157

  19. Greenfield NJ (2007) Using circular dichroism spectra to estimate protein secondary structure. Nat Protocols 1:2876–2890

  20. Guo B, Chen X, Dang P, Scully B, Liang X, Holbrook CC, Yu J, Culbreath A (2008) Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection. BMC Dev Biol 8:12

  21. Guo B, Fedorova ND, Chen X, Wan CH, Wang W, Nierman WC, Bhatnagar D, Yu J (2011) Gene expression profiling and identification of resistance genes to Aspergillus flavus infection in peanut through EST and microarray strategies. Toxins 3:737–753

  22. Hara M (2010) The multifunctionality of dehydrins: an overview. Plant Signal Behav 5:503–508

  23. Heil M (2011) Nectar: generation, regulation and ecological functions. Trends Plant Sci 16:191–200

  24. Heil M, Rattke J, Boland W (2005) Postsecretory hydrolysis of nectar sucrose and specialization in ant/plant mutualism. Science 308:560–563

  25. Hundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics 9:118

  26. Ingle RA, Schmidt UG, Farrant JM, Thomson JA, Mundree SG (2007) Proteomic analysis of leaf proteins during dehydration of the resurrection plant Xerophyta viscosa. Plant Cell Environ 30:435–446

  27. Iturriaga G, Schneider K, Salamini F, Bartels D (1992) Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco. Plant Mol Biol 20:555–558

  28. Iturriaga G, Cushman MAF, Cushman JC (2006) An EST catalogue from the resurrection plant Selaginella lepidophylla reveals abiotic stress-adaptive genes. Plant Sci 170:1173–1184

  29. Johnson WC (1999) Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins 35:307–312

  30. Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202

  31. Karlson DT, Xiang QY, Stirm VE, Shirazi AM, Ashworth EN (2004) Phylogenetic analyses in Cornus substantiate ancestry of xylem supercooling freezing behaviour and reveal lineage of desiccation related proteins. Plant Physiol 135:1654–1665

  32. Kovacs D, Kalmar E, Torok Z, Tompa P (2008) Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins. Plant Physiol 147:381–390

  33. Kram B, Bainbridge E, Perera MA, Carter C (2008) Identification, cloning and characterization of a GDSL lipase secreted into the nectar of Jacaranda mimosifolia. Plant Mol Biol 68:173–183

  34. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

  35. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  36. Liu H, Liu YZ, Zheng SQ, Jiang JM, Wang P, Chen W (2010) Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion. Planta 231:847–860

  37. Lorenz MC, Fink GR (2001) The glyoxylate cycle is required for fungal virulence. Nature 412:83–86

  38. Naqvi SMS, Harper A, Carter C, Ren G, Guirgis A, York WS, Thornburg RW (2005) Nectarin IV, a potent endoglucanase inhibitor secreted into the nectar of ornamental tobacco plants. Isolation, cloning, and characterization. Plant Physiol 139:1389–1400

  39. Nepi M, Bini L, Bianchi L, Puglia M, Abate M, Cai G (2011) Xylan-degrading enzymes in male and female flower nectar of Cucurbita pepo. Ann Bot 108:521–527

  40. Park S, Thornburg R (2009) Biochemistry of nectar proteins. J Plant Biol 52:27–34

  41. Payton P, Kottapalli K, Rowland D, Faircloth W, Guo B, Burow M, Puppala N, Gallo M (2009) Gene expression profiling in peanut using high density oligonucleotide microarrays. BMC Genomics 10:265

  42. Peres NT, Maranhão FC, Rossi A, Martinez-Rossi NM (2010) Dermatophytes: host-pathogen interaction and antifungal resistance. An Bras Dermatol 85:657–667

  43. Peumans WJ, Smeets K, Van Nerum K, Van Leuven F, Van Damme EJ (1997) Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L.) flowers. Planta 201:298–302

  44. Piatkowski D, Schneider K, Salamini F, Bartels D (1990) Characterization of 5 abscisic acid-responsive cDNA clones isolated from the desiccation-tolerant plant Craterostigma-plantagineum and their relationship to other water-stress genes. Plant Physiol 94:1682–1688

  45. Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, Beckmann JS, Silman I, Sussman JL (2005) FoldIndex©: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21:3435–3438

  46. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer EL, Eddy SR, Bateman A, Finn RD (2012) The Pfam protein families database. Nucleic Acids Res 40:D290–D301

  47. Rorat T (2006) Plant dehydrins-tissue location, structure and function. Cell Mol Biol Lett 11:536–556

  48. Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

  49. Salmi ML, Bushart TJ, Stout SC, Roux SJ (2005) Profile and analysis of gene expression changes during early development in germinating spores of Ceratopteris richardii. Plant Physiol 138:1734–1745

  50. Schneider K, Wells B, Schmelzer E, Salamini F, Bartels D (1993) Desiccation leads to the rapid accumulation of both cytosolic and chloroplastic proteins in the resurrection plant Craterostigma-plantagineum Hochst. Planta 189:120–131

  51. Srere PA (1966) Citrate-condensing enzyme-oxaloacetate binary complex. Studies on its physical and chemical properties. J Biol Chem 241:2157–2165

  52. Suksomtip M, Liu P, Anderson T, Tungpradabkul S, Wood DW, Nester EW (2005) Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction. J Bacteriol 187:4844–4852

  53. Swarbrick PJ, Huang K, Liu G, Slate J, Press MC, Scholes JD (2008) Global patterns of gene expression in rice cultivars undergoing a susceptible or resistant interaction with the parasitic plant Striga hermonthica. New Phytol 179:515–529

  54. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

  55. Thornburg RW, Carter C, Powell A, Mittler R, Rizhsky L, Horner HT (2003) A major function of the tobacco floral nectary is defense against microbial attack. Plant Syst Evol 238:211–218

  56. Tunnacliffe A, Hincha D, Leprince O, Macherel D (2010) LEA proteins: versatility of form and function. In: Lubzens E, Cerda J, Clark M (eds) Dormancy and resistance in harsh environments. Springer Berlin Heidelberg, pp 91–108

  57. Vitale A, Chrispeels MJ (1992) Sorting of proteins to the vacuoles of plant cells. BioEssays 14:151–160

  58. Wallace BA, Janes RW (2001) Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics. Curr Opin Chem Biol 5:567–571

  59. Wang ZY, Thornton CR, Kershaw MJ, Li DB, Talbot NJ (2003) The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Mol Microbiol 47:1601–1612

  60. Weigand G, Remington SJ (1986) Citrate synthase: structure, control, and mechanism. Annu Rev Biophys Biophys Chem 15:97–117

  61. Welin BV, Olson Å, Palva ET (1995) Structure and organization of two closely related low-temperature-induced dhn/lea/rab-like genes in Arabidopsis thaliana L. Heynh. Plant Mol Biol 29:391–395

  62. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 89:392–400

  63. Yang P, Li X, Wang X, Chen H, Chen F, Shen S (2007) Proteomic analysis of rice (Oryza sativa) seeds during germination. Proteomics 7:3358–3368

  64. Zha HG, Lynn Flowers V, Yang M, Chen LY, Sun H (2012) Acidic alpha-galactosidase, the most abundant nectarin in floral nectar of common tobacco (Nicotiana tabacum L.). Ann Bot 109:735–745

  65. Zuo K, Wang J, Wu W, Chai Y, Sun X, Tang K (2005) Identification and characterization of differentially expressed ESTs of Gossypium barbadense infected by Verticillium dahliae with suppression subtractive hybridization. Mol Biol 39:191–199

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Acknowledgments

We thank Professor Dorothea Bartels (University of Bonn, Germany) and Dr. Changkyun Kim (Ajou University, South Korea) for critical comments on the manuscript. This study was supported by funding for H.G. Zha from the West Light Foundation of The Chinese Academy of Sciences and from the National Science Foundation of China (grant number 31170216). H. Sun received funding from the National Natural Science Foundation and Yunnan Natural Science Foundation Joint Project (grant number U136601). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.

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Correspondence to Jing-Jiang Zhou or Hang Sun.

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Zha, H., Liu, T., Zhou, J. et al. MS-desi, a desiccation-related protein in the floral nectar of the evergreen velvet bean (Mucuna sempervirens Hemsl): molecular identification and characterization. Planta 238, 77–89 (2013). https://doi.org/10.1007/s00425-013-1876-2

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Keywords

  • Citrate synthase inhibition
  • Desiccation-related protein
  • Mucuna
  • Nectarin pcC13-62 like