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Keywords:

  • ABA-insensitive (abi) mutants;
  • ABA signaling;
  • abscisic acid (ABA);
  • Arabidopsis;
  • desiccation tolerance (DT);
  • germination;
  • re-establishment of DT;
  • seed development

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • During germination, orthodox seeds lose their desiccation tolerance (DT) and become sensitive to extreme drying. Yet, DT can be rescued, in a well-defined developmental window, by the application of a mild osmotic stress before dehydration. A role for abscisic acid (ABA) has been implicated in this stress response and in DT re-establishment. However, the path from the sensing of an osmotic cue and its signaling to DT re-establishment is still largely unknown.
  • Analyses of DT, ABA sensitivity, ABA content and gene expression were performed in desiccation-sensitive (DS) and desiccation-tolerant Arabidopsis thaliana seeds. Furthermore, loss and re-establishment of DT in germinated Arabidopsis seeds was studied in ABA-deficient and ABA-insensitive mutants.
  • We demonstrate that the developmental window in which DT can be re-established correlates strongly with the window in which ABA sensitivity is still present. Using ABA biosynthesis and signaling mutants, we show that this hormone plays a key role in DT re-establishment.
  • Surprisingly, re-establishment of DT depends on the modulation of ABA sensitivity rather than enhanced ABA content. In addition, the evaluation of several ABA-insensitive mutants, which can still produce normal desiccation-tolerant seeds, but are impaired in the re-establishment of DT, shows that the acquisition of DT during seed development is genetically different from its re-establishment during germination.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and growth conditions

The following Arabidopsis thaliana (L.) Heynh. lines were used: ecotype Columbia (Col-0); abi3-8, abi3-9, abi4-3 and abi5-7 (Nambara et al., 2002); aba2-1 (Léon-Kloosterziel et al., 1996); and an Arabidopsis ecotype C24 line expressing the RD29A::LUC (RESPONSIVE TO DESICCATION 29A) transgene (Ishitani et al., 1997). In all experiments, Arabidopsis seeds were cold stratified for 72 h at 4°C in 9-cm Petri dishes to eliminate residual dormancy. Germination assays were performed under constant white light at 22°C. Seeds of four developmental stages were used, that is, at testa rupture (stage I), at radicle protrusion (stage II), at a primary root length of 0.3–0.5 mm (stage III) and at the appearance of the first root hairs before cotyledon greening (stage IV) (Fig. 1a; adapted from Maia et al., 2011).

image

Figure 1. (a) Developmental stages of germinated Arabidopsis seeds. Developmental stages I–IV represent seeds at testa rupture (stage I), at radicle protrusion (stage II), showing a primary root of 0.3–0.5 mm in length (stage III) and at the appearance of the first root hairs before greening of cotyledons (stage IV). (b) Re-establishment of desiccation tolerance and abscisic acid (ABA) sensitivity in germinated Arabidopsis seeds. ABA sensitivity (dashed line) and survival of cotyledons (black bars), primary roots (gray bars) and seedlings (white bars). Untreated seeds at all developmental stages were completely desiccation sensitive. Values are expressed as the average of four replicates ± SE of 20–30 germinated seeds. (c) RD29A::LUC activity of germinated Arabidopsis seeds over time. Seeds at stages I (red line), II (black line) and IV (green line) were collected and treated with 5 μM ABA solution. The ‘blue line’ represents untreated samples incubated in water. RD29 promoter activity was measured using a chemiluminescence assay in the dark at 20°C. Values are expressed as the average of three replicates ± SE of 100 germinated seeds. Error bars are depicted as shading of the curves.

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Re-establishment of DT and assessment of ABA sensitivity

Re-establishment of DT and ABA sensitivity were measured in parallel in germinated wild-type and mutant seeds. To determine the re-establishment of DT, seeds at stages I, II, III and IV were dried either directly (untreated controls) or after 3 d of incubation in PEG at −2.5 MPa (treated samples). The PEG concentration used was such that it inhibited further growth of the treated germinated seeds, thus keeping them in the same developmental stage in which they were collected. After incubation, treated seeds still in stages I, II, III and IV were rinsed thoroughly in distilled water with the aid of a sieve, transferred to a new Petri dish with one dry sheet of germination paper and dehydrated for 3 d at 20°C at 32% relative humidity (RH), which was achieved over a saturated calcium chloride solution in a closed chamber. After dehydration, treated and untreated seeds were pre-humidified (100% RH) for 24 h at 22°C in the dark to avoid imbibition damage (Leopold & Vertucci, 1986). The survival of cotyledons and primary roots, 5 d after rehydration, and viable seedlings, 10 d after rehydration, was scored. Cotyledons that continued their development, became green and opened, and primary roots that were further elongated, were considered to be alive.

To determine ABA sensitivity, germinated seeds from each germination stage were transferred to 6-cm Petri dishes containing two filter papers moistened with 1 ml of 5 μM ABA. The samples were incubated at 22°C under constant light for 10 d. Germinated seeds that continued their development and possessed expanded green cotyledons were considered to be insensitive, whereas those that arrested were assessed as ABA sensitive.

Luciferase imaging and analysis

An Arabidopsis (ecotype C24) line expressing the firefly luciferase reporter gene under the control of the Arabidopsis RD29A promoter (RD29A::LUC) was used (Ishitani et al., 1997). The luciferase reporter gene under the control of the RD29A promoter was developed to screen mutants with defects in their abiotic stress signal transduction pathways (Ishitani et al., 1997). This promoter is responsive to cold, osmotic stress and ABA (Yamaguchi-Shinozaki & Shinozaki, 1993). Luciferase imaging and analysis were performed as described previously (Van Leeuwen et al., 2000) with some modifications. RD29A::LUC seeds were cold stratified for 72 h at 4°C in 9-cm Petri dishes on two layers of blue filter paper with 10 ml of 1 mM firefly d-luciferin, sodium-salt (Duchefa, Haarlem, the Netherlands). Germination was performed under constant white light at 22°C. One hundred seeds at each developmental stage were used for further analysis. RD29A promoter activity was measured by acquiring images in the dark at 17–20°C every 7 min, with exposure times of 5 min, using a Pixis 1024B camera system (Princeton Instruments, NJ, USA) equipped with a 35-mm, 1 : 1.4 Nikkor SLR camera lens (Nikon, Amsterdam, the Netherlands). Relative luminescence was analysed for each group of 100 seeds in each image as the mean grey value using ROI manager of ImageJ 1.44n software (http://imagej.nih.gov/ij/). Background subtraction was performed for all luciferase imaging experiments.

RNA extraction

Total RNA extraction was performed according to the hot borate protocol modified from Wan & Wilkins (1994), as described previously by Maia et al. (2011). At least 100 seeds were used in each extraction. RNA integrity was assessed by analysis on a 1.2% agarose gel, and RNA sample quality and concentration were additionally assessed using a Nanodrop ND-1000 (Nanodrop Technologies Inc.).

Real-time quantitative PCR (RT-qPCR) conditions

cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer's protocol. iQ-SYBR-Green-Supermix (Bio-Rad) was used for gene expression analysis on an MyIQ RT-qPCR machine (Bio-Rad). The RT-qPCR program run consisted of a first step at 95°C for 3 min, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The primers used (Supporting Information Table S1) were designed preferably in the 3′ part of the transcript. Where possible, primer pairs were designed spanning an intron–exon border. Melting curve analysis was performed after the RT-qPCR run (55–95°C with 0.5°C increments every 10 s). For all primers, a single peak was observed, confirming the synthesis of a single product. Primer efficiencies were assessed with LinReg PCR software (Ramakers et al., 2003). All primers showed efficiencies of at least 90%. Ct values were obtained from iQ5 software (Bio-Rad) (baseline subtracted, threshold value of 91 relative fluorescence units (RFU)) and analyzed with qbase+ (Biogazelle, Zwijnaarde, Belgium). Seven reference genes (Dekkers et al., 2012) were tested. Their expression stability was calculated by geNORM (Vandesompele et al., 2002; Fig. S1), and the three most stable (AT3g59990, AT2g28390 and AT3g33520 – Table S1) were used for further studies. RT-qPCR data of each gene of interest were normalized against the three selected reference genes. Finally, calibrated normalized relative quantity (CNRQ) values were exported from qbase+ and statistically analyzed with SISVAR software (Furtado, 2011).

Extraction of ABA from germinated Arabidopsis seeds

For ABA analysis, 20–50 mg of fresh untreated or treated (incubation for 3 and 7 h in −2.5 MPa PEG) seeds (stages I, II, III and IV) were frozen in liquid nitrogen and ground in a dismembrator (Mikro-dismembrator U; B. Braun Biotech International, Melsungen, Germany) at 2000 rpm for 1.5 min with the help of 0.6 cm stainless steel beads. Samples were extracted with 1.5 ml of isopropanol–acetic acid (99 : 1) containing 2.5 mM dithiothreitol and 0.025 nmol [2H6]-ABA as internal standard in a 2-ml centrifuge tube. The tubes were vortexed and sonicated for 10 min in an ultrasonic bath (Branson 3510, Danbury, CT, USA). Samples were incubated for 1 h at 4°C with shaking. After the first extraction, samples were centrifuged for 10 min (2500 g). The liquid phase was carefully transferred to a 4-ml glass vial. The pellets were re-extracted overnight with another 1.5 ml of isopropanol–acetic acid (99 : 1). The combined isopropanol–acetic acid fractions were dried in a SpeedVac centrifuge (SPD121P; Thermo Scientific, Tewksbury, MA, USA) and the residue was dissolved in 1 ml Ultra Performance Liquid Chromatography (UPLC) grade water. Samples were transferred to MAX columns (Oasis® 30 mg, 1 ml; Waters, Milford, MA, USA) previously equilibrated with 1 ml 100% methanol (HPLC supra gradient) followed by 1 ml of water. The columns/samples were washed with 1 ml UPLC grade water, followed by 1 ml 100% methanol. After washing, 1 ml methanol–2% formic acid were added to the columns in two steps of 500 μl and the flow through was collected. Samples were dried in a SpeedVac centrifuge and re-suspended in 100 μl UPLC grade water. The samples were stored at −20°C until measurement.

ABA detection and quantification by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

ABA analysis was performed by multiple reaction monitoring (MRM)-LC-MS/MS using a published protocol with some modifications (Saika et al., 2007). Analyses were carried out on a Waters Micromass Quattro Premier XE tandem mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source and coupled to an Acquity UPLC system (Waters). Chromatographic separation was achieved using an Acquity UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm; Waters), applying a water–acetonitrile gradient, starting at 0% acetonitrile for 2 min, raised to 50% (v/v) acetonitrile in 8 min, followed by a 1-min gradient to 90% (v/v) acetonitrile, which was then maintained for 0.1 min and followed by a 0.2-min gradient back to 0% acetonitrile before the next run. The column was equilibrated at this solvent composition for 2.8 min. The total run time was 15 min. The column was operated at 50°C with a flow rate of 0.4 ml min−1 and a sample injection volume was 10 μl. The mass spectrometer was operated in positive ESI mode. Nebulizer and desolvation gas flows were 50 and 800 l h−1, respectively. The capillary voltage was set at 2.7 kV, cone voltage at 10 V, source temperature at 120°C and desolvation gas temperature at 450°C. Fragmentation was performed by collision-induced dissociation with argon at 3.0 × 10−3 mbar. MRM was used for ABA quantification. Parent–daughter transitions were set according to the MS/MS spectra obtained for the standards ABA and [2H6]-ABA. Transitions were selected on the basis of the most abundant and specific fragment ions for which the collision energy (CE) was optimized. For ABA, the MRM transitions m/z 265 > 229 at a CE of 10 eV and 265 > 247 at 5 eV and, for [2H6]-ABA, the transitions m/z 271 > 234 at 10 eV and 271 > 253 at 5 eV were selected. ABA was quantified using a calibration curve with known amounts of standard and based on the ratio of the summed area of the MRM transitions for ABA to those for [2H6]-ABA. Data acquisition and analysis were performed using MassLynx 4.1 software (Waters).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

DT re-establishment in germinated Arabidopsis seeds correlates strongly with the presence of and sensitivity to ABA

We have shown previously that DT can be re-induced in germinated Arabidopsis seeds by treating them in a PEG (−2.5 MPa) solution (Maia et al., 2011). Here, we evaluated whether the window of ABA sensitivity during and after germination (Lopez-Molina et al., 2001) correlates with the time frame in which DT can be re-established. For this, sensitivity to 5 μM ABA was correlated with the ability to re-establish DT in cotyledons, primary roots and seedlings in seeds at four developmental stages during and after germination (Fig. 1a,b).

The ability of Col-0 seeds to re-establish DT during germination decreased together with their ABA sensitivity (Fig. 1b). DT could be re-established in Col-0 seeds at developmental stages I, II and III when ABA sensitivity was still high. In agreement with our previous results, Col-0 seeds at stages I and II displayed 100% of re-establishment of DT of primary roots, cotyledons and seedlings, whereas, in those at stage III, a slight reduction in DT was observed in all three structures. Low ABA sensitivity levels of germinated Col-0 seeds at stage IV correlated with a reduced ability to re-establish DT (Fig. 1b).

To further investigate the correlation between ABA sensitivity and the re-establishment of DT, we checked the promoter activity of the ABA-responsive gene RD29A during the four developmental stages (Fig. 1c). An increase in RD29A::LUC activity was measured in all seeds as quickly as 30 min after the addition of ABA. This activity reached its maximum c. 150 min after ABA incubation. Confirming our previous results on ABA sensitivity, RD29A::LUC activation also seemed to be dependent on the seed developmental stage. In relation to earlier developmental stages, seeds at stage IV showed less RD29A::LUC activity in the presence of exogenous ABA, indicating a reduction in ABA signal transduction.

Because we observed a strong correlation between ABA sensitivity and the ability of seeds to re-induce DT, we investigated whether wild-type ABA contents are needed for DT re-establishment. Therefore, we assessed the ability of ABA-deficient Arabidopsis aba2 mutant seeds to re-establish DT. Emphasizing the role of ABA in DT re-establishment, germinated seeds (stage II) of the aba2-1 mutant treated with PEG were severely compromised in their ability to re-establish DT in cotyledons, primary roots and seedlings (Fig. 2a). The ability of the aba2-1 mutant to re-establish DT was completely rescued by the addition of 1 μM ABA to the PEG solution (Fig. 2b). Interestingly, treatment with 5 μM ABA alone was also sufficient to re-induce DT in both Col-0 and aba2-1 seeds (Fig. 2c), showing that the ABA signal may substitute for the osmotic signal to re-establish DT. Although 1 μM ABA was sufficient to complement the desiccation-tolerant phenotype when applied in combination with PEG, 1 μM ABA was not sufficient when applied alone (Fig. S2). Seeds treated with 1 μM ABA did not stop developing. When applied alone, a higher ABA concentration (5 μM ABA) was needed to fully arrest development and re-induce DT.

image

Figure 2. Re-establishment of desiccation tolerance (DT) in germinated Col-0 (black bars) and aba2-1 (open bars) Arabidopsis seeds. To determine the re-establishment of DT, germinated seeds at stage II were selected and either dried directly or after 3 d of incubation in −2.5 MPa polyethylene glycol (PEG) (a), a combination of −2.5 MPa PEG + 1 μM abscisic acid (ABA) (b) or 5 μM ABA (c). Untreated control seeds were completely desiccation sensitive and are not shown. PR, primary root; Ct, cotyledons; Sdl, seedling. Values are expressed as the average of three replicates ± SE of 20–30 germinated seeds. Asterisks (**,  0.01) represent significant differences between Col-0 and aba2-1 treated seeds.

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Our results showed that Col-0 seeds at stage IV and the aba2-1 mutant had a reduced survival after DT treatment. To rule out the possibility that Col-0 seeds at stage IV and the aba2-1 mutant were killed by a possible detrimental effect caused by PEG itself, we performed an additional control experiment. In this experiment, Col-0 and aba2-1 seeds at all stages (I–IV) were incubated in −2.5 MPa PEG for 3 d and were immediately rehydrated (instead of dehydrated). All seedlings developed normally after direct rehydration following PEG treatment. This shows that the seeds were not killed by PEG, but were incapable of regaining DT by the PEG treatment (Fig. S2).

Together, these results show a fundamental role for ABA in the re-establishment of DT. Therefore, we investigated further the importance of ABA biosynthesis and signaling in this response.

Enhanced ABA content is not required for the re-establishment of DT

We investigated whether increased ABA content is necessary for DT re-establishment. Therefore, we measured the expression of ABA biosynthetic and catabolic genes and ABA content in response to PEG treatment. The expression of seven genes involved in ABA biosynthesis was investigated (NINE-CIS-EPOXYCAROTENOID DIOXYGENASENCED3, NCED5, NCED6, NCED9 and ABSCISIC ACID-DEFICIENTABA1, ABA2 and ABA3) and one gene involved in ABA degradation (CYTOCHROME P450CYP707A2) (Fig. 3a,b).

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Figure 3. (a) Diagram showing key genes involved in the regulation of abscisic acid (ABA) homeostasis in Arabidopsis thaliana (modified, with permission, from Baron et al., 2012). Genes represented in bold uppercase letters were investigated in this study. (b) Gene expression analysis by real-time quantitative PCR (RT-qPCR) represented as the calibrated normalized relative quantity (CNRQ) for ABA biosynthesis genes ABA1, ABA2, ABA3, NCED3, NCED6, NCED5, NCED9 and ABA catabolic gene CYP707A2. Gene expression is shown of untreated seeds (closed bars) and seeds treated for 5 h in polyethylene glycol (PEG) −2.5 MPa (open bars). Bars represent the mean values ± SE of three independent biological replicates. Small letters represent statistically significant differences ( 0.05) between different developmental stages within the same treatment. Asterisks (*,  0.05; **,  0.01) represent significant differences between treated and untreated seeds.

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All genes showed significant changes in their expression levels during development and after treatment (Fig. 3b). For example, NCED6 was significantly down-regulated in stages I–IV in untreated seeds. This gene was also down-regulated by PEG at stages I and II. In contrast, the expression of ABA1 and ABA2 increased significantly with seedling development in untreated seeds and, except for the expression of ABA2 in seeds at stage IV, was highly induced in PEG-treated seeds. Overall, the expression of all analyzed ABA biosynthesis genes was up-regulated in PEG-treated seeds, with the exception of NCED6. By contrast, the ABA catabolic gene CYP707A2 was down-regulated with development and in PEG-treated seeds (Fig. 3b). These expression patterns suggest that ABA content increases on PEG treatment.

To confirm our gene expression data, ABA content was determined in seeds (stages I–IV) in the presence and absence of PEG. In contrast with the observations on the expression of ABA biosynthesis genes, ABA content did not change significantly after PEG treatment when compared with untreated seeds (Fig. 4). These data suggest that either ABA is not being synthesized or is being further modified into different metabolic products. To confirm and extend our analysis, we performed a second independent experiment in a different laboratory in which ABA and its degradation and conjugation products were measured (Methods S1, Table S2). cis-Abscisic acid (ABA), trans-abscisic acid (t-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), neo-phaseic acid (neo-PA), 7′-hydroxy-ABA and abscisic acid glucose ester (ABA-GE) were measured in untreated and PEG-treated (5 and 24 h of PEG incubation) seeds at all four developmental stages. Confirming our previous results, PEG-treated seeds did not accumulate active ABA in relation to untreated seeds (Table S2). To our surprise, ABA degradation products, such as PA, DPA and neo-PA, as well as the conjugation product ABA-GE, also did not accumulate after PEG treatment, when compared with untreated seeds (Table S2). In some cases, t-ABA was detected at higher levels than ABA, but the trend was not consistent throughout stages and treatments.

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Figure 4. Abscisic acid (ABA) content of Arabidopsis seeds at different developmental stages. ABA was measured in extracts of seeds that were untreated and treated for 3 or 7 h with −2.5 MPa polyethylene glycol (PEG). Control, black bars; 3 h PEG, gray bars; 7 h PEG, white bars. Bars represent the average of three independent replicates ± SE. No statistically significant differences ( 0.05) were observed between treated and untreated seeds.

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These ABA measurements indicate that PEG-induced re-establishment of DT is not a direct consequence of increased ABA content, suggesting that another mechanism, such as enhanced ABA sensitivity or signaling, is probably involved.

Changes in the expression of ABA signaling genes and the re-establishment of DT

Our data show that ABA is important in the re-establishment of DT in germinated seeds, but it is still unclear how the ABA signaling pathway is activated and which are the main players involved. To address these questions, RT-qPCR analysis was performed on a set of genes previously described to be involved in the ABA signaling pathway. To narrow down our analysis, the expression profile of a pre-set of 38 candidate genes was checked during seed development and germination in the eFP browser at the BAR website (http://www.bar.utoronto.ca/; Toufighi et al., 2005; Table S3). Preference was given to genes that were up-regulated during seed development, concomitant with the onset of DT, and down-regulated during germination on which DT is lost (Table S3). Thirteen genes were selected for further analysis, including three ABA receptors, seven phosphatases and three TFs (Table S1).

To identify the best time points to monitor ABA signaling during the re-establishment of DT, an expression time series during PEG treatment was performed. The expression of the ABA-responsive RD29A gene was used as a reference. To obtain a first overview of RD29A promoter activity, we measured its activity using a luciferin imaging assay (Fig. S3a). Based on the luciferin activity assay results, material from germinated seeds (stage II), both untreated and incubated in −2.5 MPa PEG for 3, 7, 20, 48 and 72 h, was prepared and used for RT-qPCR analysis (Fig. S3b). Both RT-qPCR and luciferin assays demonstrated that RD29A expression was quickly up-regulated on PEG treatment (Fig. S3a,b). RD29A expression started at c. 3 h and reached its highest level 7 h after PEG incubation, after which it quickly declined after 20 h (Fig. S3a,b). Based on the expression patterns observed for RD29A under osmotic treatment, we decided to analyze the expression of the 13 selected ABA signaling genes after 5 h of PEG incubation, when ABA-responsive genes are most likely to be expressed (Fig. 5).

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Figure 5. Gene expression analysis by real-time quantitative PCR (RT-qPCR) of abscisic acid (ABA) signaling genes in germinating and germinated Arabidopsis seeds. (a) ABA receptors from the PYL/PYR/RCAR (PYRABACTIN RESISTANCE1 (PYR1)-LIKE REGULATORY COMPONENTS OF ABA RECEPTORS) family, (b) PP2Cs (TYPE 2C PROTEIN PHOSPHATASES) and (c) transcription factors. CNRQ, calibrated normalized relative quantity. Gene expression is shown for untreated seeds (closed bars) and seeds treated for 5 h in −2.5 MPa polyethylene glycol (PEG) (open bars). Bars represent the mean values ± SE of three independent biological replicates. Small letters represent statistically significant differences ( 0.05) between different developmental stages within the same treatment. Asterisks (*,  0.05; **,  0.01) represent significant differences between treated and untreated seeds.

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The expression levels of PYL7 (PYRABACTIN RESISTANCE 1-LIKE 7) and PYL9 were markedly increased after 5 h of PEG treatment (Fig. 5a). The increased expression of PYL7 and PYL9 in response to PEG indicates that these genes are possibly involved in the regulation of the re-establishment of DT in germinated seeds. No significant changes in transcript level were observed for PYL5, which suggests that either different receptor proteins are involved in the signaling for distinct environmental cues, or for different tissues, with, for example, PYL7 and PYL9 as important genes in the response to osmotic stress in germinating seeds. Apart from this, when under osmotic stress, all PYL genes showed higher gene expression after radicle protrusion (stages II, III and IV) when compared with seeds at stage I (Fig. 5a). Taken together, the enhanced expression of PYL7 and PYL9 in response to PEG suggests that increased ABA sensitivity is involved in the re-establishment of DT.

All PP2C genes evaluated were consistently up-regulated under osmotic stress at all developmental stages, but showed very diverse expression patterns during development (Fig. 5b). For instance, although HIGHLY ABA-INDUCED PP2C GENE 1 (HAI1) and ABA-INSENSITIVE 1 (ABI1) were up-regulated over time in response to osmotic stress, HAI3 and ABA HYPERSENSITIVE GERMINATION 1 (AHG1) showed the exact opposite (Fig. 5b). The expression profiles of AKT1 INTERACTING PROTEIN PHOSPHATASE 1 (AIP1), ABI2 and AHG3 did not show any clear correlation with the aptitude of DS seeds to re-establish DT. Interestingly, HAI1, ABI1 and ABI2 expression was only triggered by PEG from stage II onwards, indicating the possible existence of a link with the expression of PYL genes and a developmental switch between testa rupture and radicle protrusion.

The trends of expression found for ABI3, ABI4 and ABI5 were very similar. Their transcript abundance decreased during germination in both the absence and presence of PEG (Fig. 5c). ABI3 and ABI4 did not show significant up-regulation in PEG-treated seeds. ABI5, however, was strongly up-regulated under osmotic stress. Yet, the PEG-induced up-regulation of ABI5 decreased during development and was comparable with the ABI5 transcript levels found in untreated seeds when at stage IV (Fig. 5c). The expression pattern found for ABI5 correlated well with ABA sensitivity and the developmental window in which DT can still be re-established, which is suggestive for a function of ABI5 in the re-establishment of DT in germinated Arabidopsis seeds.

By using the expression of ABA signaling genes as an indicator of ABA sensitivity, we conclude that the developmental phase plays a crucial role in this attribute. In general, most of the changes in expression occurred with development and under PEG treatment. Remarkably, in spite of being negative regulators of ABA signaling (Gosti et al., 1999; Ma et al., 2009), all PP2Cs evaluated in this study were up-regulated in PEG-treated seeds. Our findings are in agreement with microarray expression data of young Arabidopsis seedlings exposed to osmotic, salt, drought and cold stresses, which also showed the up-regulation of most PP2C genes (Fig. S4; Toufighi et al., 2005).

ABA-insensitive and ABA-deficient mutants show reduced re-establishment of DT

We tested whether DT can be re-induced in ABA-deficient and ABA-insensitive mutants. We evaluated whether four ABA-insensitive mutants (abi3-9, abi3-8, abi4-3 and abi5-7) and one ABA-deficient mutant (aba2-1) were able to re-establish DT during and after visible germination (i.e. four developmental stages, see Fig. 1a) when treated with a −2.5 MPa PEG solution (Fig. 6).

image

Figure 6. Re-establishment of desiccation tolerance (DT) and abscisic acid (ABA) sensitivity in germinated Arabidopsis seeds. ABA sensitivity (dashed red line) and survival of cotyledons (black bars), primary roots (gray bars) and seedlings (white bars). Values are expressed as the average of four replicates ± SE of 20–30 germinated seeds.

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In spite of being fully desiccation tolerant by the end of seed maturation, all germinated mutant seeds evaluated were less competent in re-establishing DT when compared with Col-0 germinated seeds (Fig. 6). Although all the mutants were still able to recover DT when at stage I, this response was quickly lost after stage II and completely disappeared, in most cases, at stage III (i.e. abi3-9, abi3-8, abi4-3 and aba2-1; Fig. 6). The ability of Col-0 seeds to re-establish DT during germination decreased, together with their ABA sensitivity. In agreement with this, all ABA-insensitive (abi) mutants tested were less tolerant to desiccation and displayed a similar pattern of re-establishment of DT and decrease in ABA sensitivity during germination (Fig. 6). The less ABA sensitive the mutant, the quicker the decrease in its ability to recover DT with seed development. Although still sensitive to ABA in stages II and III, aba2-1 seeds were no longer able to re-establish DT (Fig. 6). This negative correlation between ABA sensitivity and DT found for this mutant indicates that, in addition to a functional ABA signaling system, the presence of a certain content of ABA is also necessary for DT re-establishment.

Although some abi mutants had similar ABA sensitivity scores, they still differed in their ability to re-establish DT (Figs 6, 7). When measuring ABA sensitivity, only expansion, greening and opening of the cotyledons were taken as parameters. Consequently, other differences in post-germination events, such as root and hypocotyl elongation, were largely neglected. Yet, these minor differences can still be seen when considering seedling appearance and fresh weight after 10 d of ABA treatment (Fig. 7). Different abi mutants that were transferred to ABA (5 μM) at the same developmental stage displayed different fresh weights and clearly showed differences in development by the end of treatment (Fig. 7). For instance, when transferred to ABA at stages III and IV, the ABA sensitivity of abi3-8 and abi3-9 was scored as 0%. However, clear differences in fresh weight and development were observed among these mutants after 10 d in ABA (Fig. 7). The abi3-8 seeds (stage III and IV) had developed further and showed visibly greater growth when compared with abi3-9 seeds transferred to ABA at the same developmental stage. These differences indicate that abi3-8 is less sensitive than abi3-9 to ABA, and could explain why, in spite of having received the same score for ABA sensitivity, abi3-8 was less competent to re-establish DT in stage III.

image

Figure 7. Abscisic acid (ABA) sensitivity during and after germination. Images depict seedlings 10 d after they were transferred to plates containing 5 μM ABA. Numbers represent ABA sensitivity as the percentage of seeds that stopped in development and did not possess expanded green cotyledons. Bar charts represent seedling fresh weight 10 d after they were transferred to Petri dishes with 5 μM ABA. Values are expressed as the average of four replicates ± SE of 20–30 germinated seeds.

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Taken together, these data confirm that ABA sensitivity plays a pivotal role in the re-establishment of DT in germinated Arabidopsis seeds. In addition, the phenotype found for the abi5-7 mutant (Figs 6, 7), in combination with the expression pattern found for this gene (Fig. 5c), highlights the importance of ABI5 in the re-establishment of DT in germinated Arabidopsis seeds.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Re-establishment of DT in germinating seeds does not require enhanced ABA content

PEG has been shown to rescue DT in germinated DS Arabidopsis seeds (Maia et al., 2011). Yet, it is not clear which mechanisms are involved and how this osmotic cue is perceived and signaled by a seed or seedling. So far, two pathways by which a plant may respond to an osmotic cue have been suggested, namely ABA-dependent and ABA-independent pathways (Yamaguchi-Shinozaki & Shinozaki, 2006). However, most frequently, experiments point towards a model dependent on ABA content-directed responses (Raghavendra et al., 2010; Fujita et al., 2011; Frey et al., 2012; Miyakawa et al., 2013). According to this view, elevated content of ABA is required to activate signaling cascades, which will ultimately lead to a stress response (e.g. re-establishment of DT). It is not clear, however, whether ABA content, synthesis, degradation and sensitivity are all important factors in the recovery of DT.

Our working hypothesis was that germinated Arabidopsis DS seeds respond to an osmotic stress (PEG) by engaging ABA biosynthesis, leading to increased ABA content. This increase in ABA content then triggers an ABA-directed stress response. However, our data suggest that increased content of ABA is not required for the re-establishment of DT in germinated Arabidopsis seeds, as we were unable to detect changes in ABA content on PEG treatment. This observation indicates that DT can be fully re-established by PEG treatment, and that this is not a direct consequence of increased ABA content. In accordance with our findings, it has been suggested that ABA accumulation is not always necessary to elicit stress responses. For instance, studies with seedlings of Salix spp. have indicated that, instead of affecting ABA content, changes in day length regulate the cessation of growth, probably by affecting ABA sensitivity (Barros & Neill, 1986). Furthermore, plants expressing a cowpea mosaic virus (CMV) factor exhibited increased drought tolerance without the over-accumulation of ABA (Westwood et al., 2013). According to these authors, CMV infection probably affected ABA signaling and/or perception instead. Together, these findings show that, next to enhancing ABA content, modulation of ABA perception and signaling can be sufficient to induce an appropriate stress response.

The content of ABA in any particular tissue in a plant is determined by the rate of biosynthesis and catabolism of the hormone (Nambara & Marion-Poll, 2005). Moreover, transport, in addition to conjugation or hydrolysis of ABA and ABA-GE, has gained increasing importance because of new discoveries in this field of research (Lee et al., 2006; Priest et al., 2006; Umezawa et al., 2010). Thus, the accumulation of ABA degradation and/or conjugation products could, in part, explain why ABA content is not increased in PEG-treated seeds. Conversely, although genes involved in ABA biosynthesis and catabolism were up- and down-regulated, respectively, suggesting that ABA content should be increased, accumulation of ABA and its degradation and conjugation products was not detected in PEG-treated seeds (Table S2). In a few cases, higher t-ABA content was detected in PEG-treated seeds, indicating an alternative route by which active ABA could be recycled. However, this trend was not consistent throughout stages and treatments.

ABA could very well be synthesized in the same ratio in which it is degraded, leading to a constant ABA balance. Furthermore, it is also possible that, instead of being synthesized as a general response, ABA may be synthesized locally in very small amounts in essential tissues or translocated from one tissue to another. In the first scenario, very small changes in ABA content would be expected and its detection would represent a bottleneck, whereas, in the case of translocation, changes in ABA content would not be expected.

The re-establishment of DT is limited to the developmental time window of ABA sensitivity and requires ABI3, ABI4 and ABI5

During germination, Arabidopsis seeds rapidly lose their DT and become sensitive to extreme drying (Maia et al., 2011). Yet, in a well-defined developmental window between the stages of testa rupture and root hair formation, seeds are still able to fully re-establish DT when submitted to a mild osmotic stress before drying (Bruggink & van der Toorn, 1995; Buitink et al., 2003; Maia et al., 2011). In addition, for other species, the re-establishment of DT is limited to a certain developmental stage after germination (Bruggink & van der Toorn, 1995; Buitink et al., 2003; Maia et al., 2011). We show here that, beyond this developmental window, the ability to re-establish DT decreases and quickly disappears, concomitant with the loss of the ability of ABA to arrest development.

The developmental time window in which seeds are able to respond to ABA has been described by Lopez-Molina et al. (2001). It was suggested that ABA, in addition to delaying germination, can also reversibly block growth during a narrow developmental time interval following germination and before the onset of vegetative growth. Within this window, ABA induces ABI5 expression and protein accumulation early in development and acts as a repressor of germination and growth. Beyond this time window, ABA is unable to induce ABI5 protein accumulation and to block growth. This window is proposed to act as a developmental checkpoint during which Arabidopsis germinating seeds can still ‘decide’, on the basis of environmental cues, to arrest development or continue germination towards seedling establishment (Lopez-Molina et al., 2001).

In our model system, ABI5 was significantly up-regulated in germinated seeds under osmotic stress at stages I, II and III (Fig. 5c). However, at stage IV, when DT cannot be induced, ABI5 expression was no longer induced by the osmotic treatment. This expression pattern matches perfectly with the developmental window in which ABA can still be perceived and DT still rescued in Arabidopsis seeds. This overlap between the induction of ABI5 expression and the re-establishment of DT suggests that this TF plays an important role in defining the developmental checkpoint that determines whether seeds can still recruit DT-related mechanisms. This was supported by the fact that mutations in this locus (i.e. abi5-7) compromise the re-establishment of DT, which was most clearly visible in stage III (Fig. 6).

In addition, genes encoding two other TFs that are important for ABA signaling, ABI3 and ABI4, are relevant in this response. These genes were not significantly up-regulated by osmotic stress, as was ABI5, but seeds mutated in these two genes clearly showed a reduced capacity to re-establish DT. The phenotypes of abi3-8, abi3-9 and abi4-3 were even stronger compared with the abi5-7 mutant, and the phenotypes were already visible at stage II. At this stage, abi5-7 was still sensitive to PEG treatment, which implies that other genes are also involved in the modulation of this temporal checkpoint. ABI5 encodes a member of the bZIP TF family (Finkelstein & Lynch, 2000b; Lopez-Molina & Chua, 2000) which comprises > 75 members (Jakoby et al., 2002). ABI5 is part of clade A of this family, which contains 13 members. Several members within this clade bind conserved cis-elements, known as ABA-responsive elements (Kim et al., 1997; Choi et al., 2000; Uno et al., 2000), and some are known to function redundantly in response to ABA and stress (Finkelstein et al., 2005; Yoshida et al., 2010). This redundancy provides an explanation of why the abi5-7 single mutant was not completely depleted in its capacity to re-establish DT.

The acquisition of DT during seed development is genetically distinguishable from DT re-establishment during germination

Many experimental models used to study DT were based on the assessment of the acquisition of DT during seed development (Blackman et al., 1992; Xu & Bewley, 1995; Nedeva & Nikolova, 1997; Black et al., 1999; Sreedhar, 2002; Illing et al., 2005). The acquisition of DT is developmentally controlled and is acquired during the seed maturation phase (Bewley et al., 2013). Seed developmental mutants that have a disrupted seed maturation program, such as lec1, lec2, fus3 and severe abi3 mutants, indeed fail to produce desiccation-tolerant seeds. Although ABA is required for normal seed development, the vast majority of ABA-insensitive and ABA-deficient mutants described to date are still able to produce completely desiccation-tolerant seeds by the end of seed maturation (Cutler et al., 2010). The only exceptions are the strong abi3 alleles or the genetic combination of the weak abi3-1 allele with the ABA-deficient aba1-1 mutant, which have an impaired capacity to acquire DT during seed maturation (Nambara et al., 1992; Ooms et al., 1993, 1994).

We used several mutants (aba2-1, abi3-8, abi3-9, abi4-3 and abi5-7) in this study. In spite of being compromised for ABA sensitivity or synthesis, all of these mutants produced desiccation-tolerant seeds at the end of seed maturation. Interestingly, at stage I, the picture seemed to be similar to that which was observed during seed development: despite the different mutated genes, DT was fully re-established. However, at stages II and III, all five mutants showed a reduced capacity to re-establish DT. The ABA-deficient aba2-1 mutant was incapable of fully recovering its DT when submitted to PEG from stage II (radicle protrusion) onwards. The inability of aba2-1 to re-establish DT implies that wild-type contents of ABA are necessary to re-establish DT in seeds at later developmental stages. In addition, in Medicago seeds, the re-establishment of DT is dependent on ABA. When an ABA biosynthesis inhibitor was applied, DT could no longer be re-established (Buitink et al., 2003). In the same report, it was also shown that the application of exogenous ABA fully rescued the DT phenotype, similar to the observations in Arabidopsis seeds. The observation that the window in which DT can be re-established is much narrower in abi mutants than in the wild-type suggests that ABI TFs are very relevant to the re-establishment of DT in Arabidopsis seeds, and that they modulate the window in which DT can be re-induced.

These observations show that the acquisition of DT during seed development is different from the re-establishment of DT in geminating seeds. From our results, two hypotheses can be formulated. First, there are (largely) distinct pathways involved in the induction of DT during seed development and during germination. Considering the fact that seeds of a strong abi3 allele or the abi3-1 aba1-1 double mutant indeed lacked DT, it is evident that ABI3 acts in both pathways. Second, the pathways that induce DT also involve ABA, ABI4 and ABI5, but their function remains unseen (when testing the mutant alleles), because of additional redundant factors present during seed development. Contrary to the seed developmental DT network, the network in the germinating seed needs to be rapidly induced de novo on osmotic stress, and may therefore lack sufficient redundancy to counteract mutations in its components. During the transitions from an embryonic to a post-germinative transcriptional program, dramatic changes occur, and thus such redundant factors may no longer be expressed, thereby revealing a role for ABA, ABI4 and ABI5 in the acquisition of DT.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Special thanks go to Kerstin Guhl for help with the ABA measurements, and to Ralph Bours and Dr Sander van der Krol for help with the luciferin assays. This work was supported by the ‘Coordenação de Aperfeiçoamento de Pessoal de Nível Superior’ (CAPES, Brazil) (J.M.) and by the Netherlands Organization for Scientific Research (NWO) (B.J.W.D.).

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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Fig. S1 Expression stability of reference genes.

Fig. S2 Survival of germinated Arabidopsis Col-0 and aba2-1 seeds incubated in polyethylene glycol (PEG) and directly rehydrated, and re-establishment of desiccation tolerance in germinated Arabidopsis Col-0 and aba2-1 seeds incubated in 1 μM abscisic acid (ABA) solution in the absence of PEG.

Fig. S3 RD29A gene expression and RD29A::LUC activity of germinated Arabidopsis seeds in response to polyethylene glycol (PEG) incubation.

Fig. S4 Heat maps showing the expression of 13 abscisic acid (ABA) signaling genes in the shoots and roots of young seedlings under different abiotic stresses.

Table S1 Genes and primer sequences used in this study

Table S2 Quantification of abscisic acid (ABA) and ABA metabolites in Arabidopsis seeds at testa rupture (I), at radicle protrusion (II), when showing a primary root of 0.3–0.5 mm in length (III) and at the appearance of the first root hairs before greening of cotyledons (IV) after 5 and 24 h of incubation in polyethylene glycol (PEG)

Table S3 Details of the heat maps displaying the gene expression profile of a pre-set of 38 candidate genes during seed development and germination according to the Bio-Array Resource database, eFP browser at the BAR website (http://www.bar.utoronto.ca)

Methods S1 Plant material, abscisic acid (ABA) extraction and quantification by HPLC-ESI-MS/MS.