Overview History Project Summary
The proteins of the translational apparatus: An Achilles' heel of seed longevity.
Figure 1: In dehydrated seeds, there exists a protein- and mRNA-store that is subjected to a loss of water but is rehydrated upon imbibition. Should the damage incurred by: 1) the stored proteome, result in loss of function of a substantial portion of the proteins within a particular pathway there is the possibility that they may be replaced by either; 2) translation from the stored transcriptome or; 3) by de novo transcription and translation. However, loss of a substantial portion of those proteins essential for translation (colored purple and associated with the ribosome above) prevents their own replacement from either stored- or de novo-produced transcripts.
Phage cDNA display of normalized mature seed libraries panned over recombinant PIMT1 from Arabidopsis in the presence of: 1) AdoMet; 2) no co-factor or; 3) the potent PIMT inhibitor, S-adenosyl homocysteine was successful identifying proteins of the translational apparatus as highly susceptible to, and/or vital to protect from, isoAsp formation . This has led to the hypothesis that, in seeds, the complete loss of function of stored proteins in all pathways except that of the translational apparatus, can be rectified by de novo translation of stored-, or newly transcribed-mRNA . Hence, the proteins of the translational apparatus might be viewed as an Achilles’ heel of seed longevity in storage (Fig. 1).
One of the PIMT targets recovered from the biopans over PIMT1 and not directly involved in translation was the SEED MATURATION PROTEIN1 (At3G12960), a homolog to the soybean putative LEA protein, GmPM28 (Glyma08G18400). This was intriguing as SMP1, with a presumptive protective role, must need protection from isoAsp formation by PIMT1, possibly to retain its function, forming part of an interactive network of protein protective mechanisms extant in seeds. Furthermore, seeds from T-DNA insertional mutants of this LEA protein gene, in two different ecotypes, were incapable of entering secondary dormancy when exposed to supraoptimal (40ºC) germination temperatures for several days prior to being placed at permissive temperatures (25ºC). Recombinant SMP1 and recombinant GmPM28 were used in phage display screens to identify their assumed endogenous client protein (ECP) targets. The ECPs identified by either LEA protein bait did not have a single target protein in common with the PIMT1 screens and yet, proteins involved in translation were again prominent in the hits . Additionally, there were some Arabidopsis protein fragments that were retrieved by both recombinant proteins . These results suggest that the proteins of the translational apparatus are indeed important to protect from dysfunction upon loss of water during maturation desiccation either directly, by LEA protein interaction, or indirectly, by repair of isoAsp formation upon subsequent imbibition.
A second collaborative project used a recombinant basic Helix-Loop-Helix (bHLH) transcription factor (tf) PHYTOCHROME INTERACTING FACTOR1 (PIF1) as a phage display bait revealed that one specific LEA protein (At2G35300) persistently bound PIF1. Why would the PIF1 tf associate with a LEA protein? This led to the hypothesis that the LEA protein might be safeguarding the proteomic memory of environmental conditions the seed has experienced thus far following imbibition. The majority of studies have demonstrated that, if hydration is sufficiently long to allow germination to progress but prior to the protrusion of some part of the embryo from the seed, there is no reset upon dehydration (i.e. the seed retains the capacity, upon re-hydration, to recommence germination at the point at which it was dehydrated (reviewed in )). Desiccation after a period of imbibition during which important environmental cues had been perceived (and the transcriptome/proteome altered accordingly) but prior to radicle protrusion, would expose the proteome, and the integrated environmental information it represents, to deleterious conditions. This eventuality necessitates protective mechanisms are put in place to ensure the dehydrated proteins retain their function so that germination can resume at the appropriate point at which it left off once the seeds are re-hydrated. Dubrovsky  referred to the capacity of seeds to resume germination from the point at which they had progressed prior to dehydration as the “Seed hydration memory”.
The concept of a seed hydration memory and results obtained using phage display has alerted us to an expanded role for LEA proteins, from that of allowing seeds to survive loss of water during maturation desiccation or after imbibition [5, 6], to now include safeguarding environmental cues, acquired during the imbibed period and embodied in a desiccation-sensitive proteome. A LEA protein apparently protects PIF1, a tf produced after imbibition and responsible for the downstream consequences of light perception through the phytochromes. Additionally, the dysfunction of some heat labile molecule, when not protected by SMP1, results in a seed that cannot “remember” the supra-optimal temperature it has experienced and behaves inappropriately, completing germination immediately when removed to 25˚C rather than entering secondary dormancy .
Thus has my lab been drawn into a survey of representative LEA proteins for their endogenous client proteins, should they exist, in two different species, Arabidopsis thaliana and Glycine max.
1. Chen, T., N. Nayak, S.M. Majee, J. Lowenson, K.R. Schafermeyer, A.C. Eliopoulos, T.D. Lloyd, R. Dinkins, S.E. Perry, N.R. Forsthoefel, S.G. Clarke, D.M. Vernon, Z.S. Zhou, T. Rejtar, and A.B. Downie, Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferase1 identified using phage display and biopanning. J Biol Chem, 2010. 285(48): p. 37281-37292.
2. Kushwaha, R., T.D. Lloyd, K.R. Schäfermeyer, S. Kumar, and A.B. Downie, Identification of Late Embryogenesis Abundant (LEA) protein Putative Interactors Using Phage Display. International Journal of Molecular Sciences, 2012. 13 (Special issue on Phage Display)(6): p. 6582-6603.
3. Hegarty, T.W., The physiology of seed hydration and dehydration, and the relation between water stress and the control of germination: a review. Plant, Cell and Environment, 1978. 1(2): p. 101-119.
4. Dubrovsky, J.G., Seed hydration memory in Sonoran desert cacti and its ecological implication. American Journal of Botany, 1996. 83(5): p. 624-632.
5. Buitink, J., B.L. Vu, P. Satour, and O. Leprince, The re-establishment of desiccation tolerance in germinated radicles of Medicago truncatula Gaertn seeds. Seed Science Research, 2003. 13(4): p. 273-286.
6. Maia, J., B.J. Dekkers, N.J. Provart, W. Ligterink, and H.W. Hilhorst, The re-establishment of desiccation tolerance in germinated Arabidopsis thaliana seeds and its associated transcriptome. PLoS One, 2011. 6(12): p. e29123.