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The seed is a key structure in the plant life cycle which aids the dispersal and survival of the species. Traits such as seed dormancy and desiccation tolerance (DT) are important in this respect. DT can be regarded as the ability of an organism to tolerate extreme water loss, whilst retaining structural integrity and viability. DT is common in most seeds, but not in vegetative tissues of angiosperms, except for a few so-called resurrection plants. Seeds which are able to withstand extreme drying to water contents below 0.1 g H2O g−1 dry weight are termed ‘orthodox’ (Bewley & Black, 1994; Bewley et al., 2013).
In orthodox seeds, DT is acquired during the maturation phase, which involves a complex regulatory network (Jia et al., 2013; Verdier et al., 2013). In Arabidopsis, seed maturation is controlled by master regulators, which interact in a complex manner, and include the CCAAT-box binding factor LEAFY COTYLEDON (LEC1) and the three B3 domain-containing proteins ABSCISIC ACID INSENSITIVE (ABI3), FUSCA (FUS3) and LEC2. Collectively, these are also known as the LAFL network. This network exerts tight control of developmental processes to ensure normal seed development and maturation, including the acquisition of DT, by affecting the expression of downstream targets, including other transcription factors (TF), hormonal pathways and the expression of SEED STORAGE PROTEIN (SSP) and LATE EMBRYOGENESIS ABUNDANT (LEA) genes (To et al., 2006; Gutierrez et al., 2007; Santos-Mendoza et al., 2008; Jia et al., 2013).
As soon as dry seeds are rehydrated, they quickly lose their DT and thus become desiccation sensitive (DS) again (Bewley & Black, 1994; Bewley et al., 2013). However, in a well-defined developmental window, it is possible to rescue DT in germinating seeds by the application of a mild osmotic stress before drying (Bruggink & van der Toorn, 1995; Buitink et al., 2003; Maia et al., 2011). This model of the re-establishment of DT in germinated seeds has already been used to investigate DT in several species (Bruggink & van der Toorn, 1995; Buitink et al., 2003; Vieira et al., 2010; Maia et al., 2011). In these studies, germinated seeds were exposed to various concentrations of polyethylene glycol (PEG) or PEG in combination with exogenous abscisic acid (ABA) or ABA biosynthesis inhibitors. This model of the re-establishment of DT in germinated seeds has also been used to study the transcriptome related to DT in the model plants Medicago truncatula and Arabidopsis thaliana (Buitink et al., 2006; Maia et al., 2011). In these studies, a marked enrichment of TF binding sites containing ABA-responsive elements among the promoters of the most highly up-regulated DT-associated genes was reported, implying that ABA plays a crucial role in their regulation (Buitink et al., 2006; Maia et al., 2011).
ABA is a phytohormone known to be a central regulator of plant development and responses to environmental stresses. To date, over 100 loci have been identified as being involved in ABA signaling (Cutler et al., 2010), including the ABA-insensitive loci abi1, abi2, abi3 (Koornneef et al., 1984), abi4 and abi5 (Finkelstein, 1994). ABI1 and ABI2 encode type 2C protein phosphatases (PP2Cs) and ABI3, ABI4 and ABI5 are TFs of the B3, APETALA2 (AP2) and basic leucine zipper (bZIP) classes, respectively (Giraudat et al., 1992; Leung et al., 1997; Finkelstein et al., 1998; Finkelstein & Lynch, 2000b; Lopez-Molina & Chua, 2000). ABI5, a TF that has an important role in the response to exogenous ABA during germination, defines a narrow developmental window, following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth (Lopez-Molina et al., 2001). In Arabidopsis seeds, ABA regulates ABI5 accumulation and activity during a limited window between 12 and 48 h of germination. Dry seeds of abi5 mutants show reduced transcript levels of ABA-responsive genes, and it has been hypothesized that ABI5 is necessary to bring germinated embryos into a quiescent state under drought stress, thereby protecting young seedlings from the loss of water (Finkelstein & Lynch, 2000a; Lopez-Molina et al., 2001).
The ABI proteins are part of a recently discovered cascade of events involving ABA receptors, protein phosphatases and protein kinases (Ma et al., 2009; Park et al., 2009). The core of this pathway consists of three protein families: the PYR/PYL/RCAR receptor family consisting of PYRABACTIN RESISTANCE1 (PYR1)-LIKE REGULATORY COMPONENTS OF ABA RECEPTORS, the TYPE 2C PROTEIN PHOSPHATASES (PP2Cs) and the SUCROSE-NON-FERMENTING KINASE1-RELATED PROTEIN KINASE2 (SnRK2) family (Umezawa et al., 2010). Together, these three protein families form a double-negative regulatory pathway. In the absence of ABA, the PP2Cs inactivate SnRK2s by dephosphorylation (Umezawa et al., 2009). Conversely, when ABA is present, it binds to the PYL/PYR/RCAR receptors, thus creating a complex which interacts with the PP2Cs. Via this interaction, the dephosphorylation of the SnRK2s by the PP2Cs is inhibited (Ma et al., 2009; Park et al., 2009). The active kinases subsequently phosphorylate different proteins, including membrane proteins and TFs (e.g. ABI5), eventually leading to an ABA response.
In this study, we investigated the role of ABA in the loss and re-establishment of DT in the model plant Arabidopsis using physiological assays, gene expression analysis and hormone measurements. We found that ABA is essential to re-establish DT by an osmotic treatment in germinated Arabidopsis seeds. Surprisingly, the re-establishment of DT seemed not to be dependent on enhanced ABA content, but was more likely driven by the modulation of ABA sensitivity. Finally, several ABA-deficient and ABA-insensitive mutants, which produce normal desiccation-tolerant seeds, were impaired in their ability to re-establish DT during germination, suggesting that the acquisition of DT during seed development is genetically distinct from the re-establishment of DT during germination.