Comparative Analysis of the Heat Stable Proteome of Radicles of Medicago truncatula Seeds during Germination Identifies Late Embryogenesis Abundant Proteins Associated with Desiccation Tolerance

Abstract

A proteomic analysis was performed on the heat stable protein fraction of imbibed radicles of Medicago truncatula seeds to investigate whether proteins can be identified that are specifically linked to desiccation tolerance (DT). Radicles were compared before and after emergence (2.8 mm long) in association with the loss of DT, and after reinduction of DT by an osmotic treatment. To separate proteins induced by the osmotic treatment from those linked with DT, the comparison was extended to 5 mm long emerged radicles for which DT could no longer be reinduced, albeit that drought tolerance was increased. The abundance of 15 polypeptides was linked with DT, out of which 11 were identified as late embryogenesis abundant proteins from different groups: MtEm6 (group 1), one isoform of DHN3 (dehydrins), MtPM25 (group 5), and three members of group 3 (MP2, an isoform of PM18, and all the isoforms of SBP65). In silico analysis revealed that their expression is likely seed specific, except for DHN3. Other isoforms of DNH3 and PM18 as well as three isoforms of the dehydrin Budcar5 were associated with drought tolerance. Changes in the abundance of MtEm6 and MtPM25 in imbibed cotyledons during the loss of DT and in developing embryos during the acquisition of DT confirmed the link of these two proteins with DT. Fourier transform infrared spectroscopy revealed that the recombinant MtPM25 and MtEm6 exhibited a certain degree of order in the hydrated state, but that they became more structured by adopting a helices and b sheets during drying. A model is presented in which DT-linked late embryogenesis abundant proteins might exert different protective functions at high and low hydration levels.

Spatiotemporal Seed Development Analysis Provides Insight into Primary Dormancy Induction and Evolution of theLepidium DELAY OF GERMINATION1Genes

“…2 and 7), we captured in type I infructescences stages prior to and at the onset of seed germinability until full premature germination (prior to dormancy induction), and in types II and III infructescences we captured all stages prior to, during, and after the induction of primary dormancy, including the full premature germination stage. To assign these stages to events during seed development and maturation on the molecular level, we analyzed the expression patterns of well-known marker proteins including oleosin (seed storage accumulation), EM6 (seed maturation), and dehydrin (desiccation tolerance; Close et al, 1993;Huang, 1996;Bies et al, 1998;Crowe et al, 2000;Ruuska et al, 2002;Boudet et al, 2006;Siloto et al, 2006;Leprince and Buitink, 2010). Oleosin accumulation during L. papillosum seed development was enhanced with the onset of premature germinability and early maturation in type I and II infructescences.…”

“…kD indicates the protein molecular mass marker ladder. [See online article for color version of this figure.] assign these protein patterns of LepaDOG1 expression to developmental stages within the L. papillosum infructescence, we used monoclonal antibodies directed against oleosin and polyclonal antibodies against EM6 (for Early Met labeled) and dehydrin; the expression abundance of these three proteins is indicative for seed storage accumulation, maturation, and the induction of desiccation tolerance, respectively (Close et al, 1993;Crowe et al, 2000;Boudet et al, 2006;Siloto et al, 2006). In support of the finding that LepaDOG1 is already abundant in immature FOs, abundant LepaDOG1 expression preceded oleosin expression and, therefore, occurs early during seed development already at the filling stage (Fig.…”

<b>‘Desiccation tolerance of <i>Rhamnidium elaeocarpum</i> Reissek (Rhamnaceae) seeds

“…These free radicals can react with hydrogen peroxide to produce singlet oxygen and hydroxyl radicals (OH-) that are toxic to cells and can damage cellular constituents, such as proteins, DNA and membranes. Free radical removers accumulate because the systems do not function as effectively in dehydrated bodies (BOUDET et al, 2006). Desiccation tolerance may be related, at least in part, to the cell's ability to sequester reactive oxygen species and therefore avoid deleterious effects such as lipid peroxidation, which is caused by these reactive species (VERTUCCI; FARRANT, 1995).…”

“…The LEA proteins are generally able to protect other proteins or membranes, similar to the actions of sugars, acting as water replacement molecules (BOUDET et al, 2006). LEA proteins are widely distributed among species of monocots and dicots, and because of their amphipathic nature, these proteins are able to inhibit the denaturation of macromolecules and stabilise intracellular structures under stress conditions because they are associated with the desiccation tolerance of the seeds (BLACKMAN et al, 1995;CLOSE, 1997).…”

Physiological and biochemical changes during the loss of desiccation tolerance in germinating Adenanthera pavonina L. seeds

“…In addition, the disappearance of LEA proteins and heat-stable proteins, was related with loss of desiccation tolerance in radicles of M. truncatula (Boudet et al 2006) and seeds of soybean (Blackman et al 1991), respectively.…”