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Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display
Department of Horticulture, University of Kentucky, Lexington, KY 40546-0312, USA
Seed Biology Group, University of Kentucky, Lexington, KY 40546-0312, USA
* Author to whom correspondence should be addressed.
Received: 29 February 2012; in revised form: 7 April 2012 / Accepted: 17 May 2012 / Published: 29 May 2012
Abstract: Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 °C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom of microtiter plates. Maintenance of a biological function for both recombinant LEA proteins when immobilized encouraged a biopanning approach to screen for potential protein interactors. Phage display with two Arabidopsis seed, T7 phage, cDNA libraries, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed seeds, were used in biopans against recombinant SMP1 and GmPM28. Phage titer increased considerably over four rounds of biopanning for both LEA proteins, but not for BSA, at both 25 and at 41 °C, regardless of the library used. The prevalence of multiple, independent clones encoding portions of specific proteins repeatedly retrieved from different libraries, temperatures and baits, provides evidence suggesting these LEA proteins are discriminating which proteins they protect, a novel finding. The identification of putative LEA-interacting proteins provides targets for reverse genetic approaches to further dissect the induction of secondary dormancy in seeds in response to heat stress.
Keywords: secondary dormancy; seed germination; heat stress; LEA proteins
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Kushwaha, R.; Lloyd, T.D.; Schäfermeyer, K.R.; Kumar, S.; Downie, A.B. Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display. Int. J. Mol. Sci. 2012, 13, 6582-6603.
Kushwaha R, Lloyd TD, Schäfermeyer KR, Kumar S, Downie AB. Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display. International Journal of Molecular Sciences. 2012; 13(6):6582-6603.
Kushwaha, Rekha; Lloyd, Taylor D.; Schäfermeyer, Kim R.; Kumar, Santosh; Downie, Allan Bruce. 2012. "Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display." Int. J. Mol. Sci. 13, no. 6: 6582-6603.