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.
Highlights
The Late Embryogenesis Abundant (LEA) proteins were first identified [1], and named [2], from studies of cotton seed proteins, and homologues have been identified in a variety of organisms within and outside the plant kingdom in the ensuing 30 years [3,4]
Assuming that the heat-labile, protected component was a protein, and using recombinant SEED MATURATION PROTEIN1 (SMP1) and its soybean LEA protein homologue (GmPM28) as bait, phage display cDNA libraries were used to pan for proteins to which the LEA proteins would bind at 25 and 41 °C to identify (a) protein(s) involved in inducing secondary dormancy in Arabidopsis thaliana due to heat stress
(a) Heated E. coli lysates of SMP1; (b) Heated fraction 15 GmPM28 recombinant protein; (c) Both SMP1 and GmPM28 were recovered from lysed E. coli and the hexahistidyl tagged proteins purified on a nickel-charged column
Summary
The Late Embryogenesis Abundant (LEA) proteins were first identified [1], and named [2], from studies of cotton seed proteins, and homologues have been identified in a variety of organisms within and outside the plant kingdom in the ensuing 30 years [3,4]. Assuming that the heat-labile, protected component was a protein, and using recombinant SMP1 and its soybean LEA protein homologue (GmPM28) as bait, phage display cDNA libraries were used to pan for proteins to which the LEA proteins would bind at 25 and 41 °C to identify (a) protein(s) involved in inducing secondary dormancy in Arabidopsis thaliana due to heat stress. The recurrent selection of at least three of these proteins (all of unknown function) provides potential targets to assess for involvement in secondary seed dormancy This is the first time LEA proteins have been documented to be selective for the proteins to which they possibly bind, which is a novel finding for potential protein interactors akin to that described for the mitochondrial localized LEA protein for membrane phospholipids of a mitochondrial composition [24]
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