title: Identification of Late Embryogenesis Abundant (LEA) protein putative interactors using phage display.
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.
authors: Department of Horticulture, University of Kentucky, Lexington, KY 40546-0312, USA.|Seed Biology Group, University of Kentucky, Lexington, KY 40546-0312, USA.
authors: Rekha
authors: R
authors: Kushwaha
authors: Department of Horticulture, University of Kentucky, Lexington, KY 40546-0312, USA.|Seed Biology Group, University of Kentucky, Lexington, KY 40546-0312, USA.
authors: Taylor D
authors: TD
authors: Lloyd
authors: Department of Horticulture, University of Kentucky, Lexington, KY 40546-0312, USA.|Seed Biology Group, University of Kentucky, Lexington, KY 40546-0312, USA.
authors: Kim R
authors: KR
authors: Schäfermeyer
authors: Department of Horticulture, University of Kentucky, Lexington, KY 40546-0312, USA.|Seed Biology Group, University of Kentucky, Lexington, KY 40546-0312, USA.
authors: Santosh
authors: S
authors: Kumar
authors: Department of Horticulture, University of Kentucky, Lexington, KY 40546-0312, USA.|Seed Biology Group, University of Kentucky, Lexington, KY 40546-0312, USA.
authors: Allan Bruce
authors: AB
authors: Downie
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D018869:Heat-Shock Response; D006358:Hot Temperature; D019151:Peptide Library; D057445:Plant Dormancy; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D000072260:RNA Splicing Factors; D011994:Recombinant Proteins / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry; D013025:Glycine max / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D018076:DNA, Complementary; D019151:Peptide Library; D010940:Plant Proteins; D000072260:RNA Splicing Factors; D011994:Recombinant Proteins; C507064:SMP1 protein, Arabidopsis; D012709:Serum Albumin; C036483:T-DNA; D014157:Transcription Factors; D005954:Glucosephosphate Dehydrogenase