Physiology of seed germination after prolonged storage
Since after 63 weeks of storage in all conditions germination percentages at 25 °C were always very low or null (Figure 1), we investigated whether seeds stored for this duration were died or deeply dormant. Germination at 15 °C, the optimal temperature for Arabidopsis seeds, revealed that seeds stored at 75 and 85 % RH were died after prolonged storage at all temperatures (Figure 4), which was also confirmed by the absence of staining in the presence of tetrazolium chloride (TZ) (Table 2). Lower RHs maintained seed viability when seed were stored at 10 or 15 °C, since seeds all germinated at 15 °C and almost all developed red staining in presence of TZ (Table 2). This revealed a process of secondary dormancy induction. In contrast, increasing temperature of storage to 20 and 25 °C lead to a decrease in seed viability, as shown Figure 4 and by lower staining of tissues by TZ (Table 2). At these temperatures only RHs close to 50 % allowed seeds survival (Figure 4 and Table 2). The use of several methods known to alleviate dormancy, such as cold stratification, GA and ethylene treatments permitted seeds stored for 63 weeks to germinate at 25 °C (Table 2) and confirmed the data shown in Figure 4.
To gain more insights about the changes in dormancy level we investigated the expression of genes known to be involved in the regulation of primary and secondary dormancy in freshly harvested seeds (D1), and seeds stored for 7 (ND) and 63 (D2) weeks at 56 % RH and 20 °C. Gene expression Seeds Values of Q10 at the seed moisture content (gH2O g dw-1).
was evaluated in dry seeds and in seeds imbibed for 24 h at 25 °C in the dark, using real-time reverse transcription-PCR (RT–PCR) (Supplementary Table S1). The abundance of all genes studied did vary significantly when assessed in dry seeds (Figure 5). The expression of NCED3, NCED6, NCED9 and ABI5 followed the same pattern. It decreased significantly in non-dormant imbibed seeds (i.e. seeds after-ripened for 7 weeks) but increased markedly in seeds imbibed after prolonged storage (i.e. 63 weeks). In contrast, abundance of CYP707A2, Ga20ox4, Ga3ox1 and SLP2 transcripts in imbibed seeds was higher in seed that had been for 7 weeks than in freshly harvested seeds. After 63 weeks of storage, expression of these genes in imbibed seeds decreased to the same level as the one found in freshly harvested seeds, excepted for CYP707A2 whose expression did not decrease significantly. At last, expression of Ga2ox2 was similar in all imbibed samples.
Primary dormancy alleviation dependency on temperature and RH
Studying the role of storage conditions of primary dormant Arabidopsis seeds revealed the complex relationship between seed MC and temperature for the control of dormancy release (Figure 1). Whatever the genotype and the conditions of storage used here, total or partial dormancy release occurred within 7 weeks, but its kinetics greatly depended on RH and temperature. As already demonstrated (see Finch Savage and Leubner Metzger, 2006) we show that dormancy alleviation is favoured at intermediates RHs, i.e. close to 50 %, but that it is slowed for extremes RHs, i.e. in very dried seeds or in high air humidity. We also show that dormancy release is faster at 20 °C, which allows proposing that the optimal combination for alleviation of dormancy in Arabidopsis seeds is 20 °C and 56 % RH. From the sorption isotherms shown Figure 2, one can determine the corresponding optimal MC which is close to 0.05 g H2O g dw-1 at 20 °C (corresponding to 56 % RH). This MC fell within the water-binding region 2 of sorption isotherms, which corresponds to weakly bound waterbut nevertheless limits biochemical reactions. Water sorption isotherms shown here displayed the classical sigmoidal shapes and the RHs at which the transitions occurred from different water-binding regions were roughly similar to those found in other ecotypes of Arabidopsis (Hay, et al. 2003) or in oily seeds such as sunflower (Bazin et al., 2011).
The results presented here reveal a tight and complex relationship between temperature and RH in the kinetics of after-ripening, and Figure 3 demonstrates that the higher the seed MC, the higher the
optimal temperature for after-ripening. For example, similar kinetics of seed dormancy alleviation can be obtained at 10 °C or 25 °C if seed MC is close to 0.04 or 0.08 g H2O g dw-1, respectively. Thermodynamic component of seed dormancy release can be appreciated using Arrhenius plots (Figure 3B, D, F) and calculation of Q10 values (Table 1). The effect of temperature on enzyme activities is well described by the Arrhenius activation energy, which represents the effect of temperature on the catalytic rate constant, and it is widely admitted that rates of biological activity increase exponentially with temperature (Brown et al., 2004). However dry seeds are a case species in which absence of free water prevents active metabolism. Arrhenius plots shown Figure 3B, 3D and 3F allow proposing the seed MC value of 0.06 g H2O g dw-1 as being critical for governing the nature of the mechanisms leading to seed dormancy release. Indeed, below 0.06 g H2O g dw-1 dormancy release in Arabidopsis was associated with negative activation energies, as shown by the negative slot of the plots (Figure 3B, D, F). The rate of dormancy release thus decreased with increasing temperature. In sunflower seeds it was also found that dormancy alleviation during after-ripening was associated with negative activation energies, when seed MC was below 0.1 g H2O g dw-1 (Bazin et al., 2011). For biological models, negative activation energies have been shown in mammalian cell lines exposed to hypothermia, which causes chilling injury and killing and they have been attributed to gel-to-liquid crystalline transition of lipids and protein denaturation (Muench et al., 1996). However, in the present study the major determinant was MC, but not temperature, since negativ and positive activation energy occurred within the range 10/25 °C, as also shown by the Q10 values which are clearly below 1 at lowest MC (Table1). In plants, metabolic rates double with every 10 °C increase in temperature usually giving Q10 values close to 2 (Criddle et al., 1994). This was the case here but only when seed MC was higher than 0.12 g H2O g dw-1 (Table 1). Altogether these data therefore suggest that dormancy alleviation at low MC does not involve metabolic activities, contrary to what occur when seed MC increases and when Q10 values are close to 2 (Table 1). As suggested for dormancy alleviation of sunflower seeds, dormancy release of Arabidopsis may be achieved by distinct mechanisms as a function of seed MC. At low MC, we propose that seed dormancy release occurs through non-enzymatic oxidative reactions which are known to be associated with negative activation energies (Cheney, 1996; Cunningham et al., 1999; Olson et al., 2002; Bazin et al., 2011).
Elementary reactions exhibiting negative activation energies are reactions for which increasing the temperature reduces probability of the colliding molecules capturing one another. This is in agreement with the concept of “oxidative window” which postulates that a certain oxidative load is needed to break dormancy (Bailly et al., 2008). When seed MC is above ca 0.12 g H2O g dw-1, metabolic reaction are likely to occur and to participate to dormancy alleviation. Then, the activation energy (given by the slope of the plots) increases when seed MC increases thus showing that changes in temperature have a significant effect on the rate of dormancy alleviation. At last, between 0.06 and 0.12 g H2O g dw-1, the activation energy is low within the studied temperature range which shows that changes in temperature have then little effect on the rate of dormancy alleviation.
Polysome profiling of dormant and non-dormant Arabidopsis seeds
At harvest, Arabidopsis seeds were dormant since they only germinated to ca 25 % at 25 °C in darkness (Figure 1A). After 3 weeks of storage at 56 % relative humidity (RH) and 20 °C, dormant seeds germinated to 75 % and after 4 weeks of after-ripening under these conditions seeds became non-dormant since they fully germinated at 25 °C (Figure 1A). In non-dormant seeds, germination at 25 °C in darkness did not begin before 2 days and was completed after 7 days (Figure 1A). At 15 °C both dormant and non-dormant seeds fully germinated after 4 days (data not shown, see Leymarie et al., 2012). In the present study, we choose data points at durations lower than 24 h at 25 °C because they corresponded to time points during germination sensu stricto, i.e. before radicle protrusion and allowed the comparison between dormant and non-dormant seeds in a similar physiological state. Sucrose gradient separation (Mustroph et al., 2009) was used to purify the polysomal fraction of RNA at regular time points during seed imbibition at 25 °C. The absorbance profiles of ribosome complexes showed that dry seeds contained a large amount of 80S monosomes and few or no polysomes (Figure 1B). In dormant seeds, early imbibition, from 3 to 6 h, was first associated with a decrease in the amount of 80S monosomes but was followed by an increase of this peak after 16 and 24h (Figure 1B).After 24 h of imbibition, the amount of 80S was significantly higher in non-dormant than in dormant seeds (Figure 1C). However, the amount of polysomal mRNAs, which increased during seed imbibition, was similar in dormant and non-dormant-seeds (Figure 1C).The amounts of polysomal components, estimated by measuring heights of peaks relative to the baseline, confirmed the visual evaluation of absorbance profiles (Supplemental Table S1). We determined whether the 80S monosomes that accumulated in dry seeds and after 24 h of imbibition were associated with mRNA. Eukaryotic ribosomes that are not associated with mRNA dissociate into subunits in solutions of high ionic strength (0.8 M KCl) (Martin, 1973) while free ribosomes are dissociated in these solutions. In dry dormant seeds, or imbibed for 24 h, the height of 80S peak was similar with KCl 0.2 M or 0.8 M (Supplemental Figure S1, A and B) which showed that monosomes were associated with RNA. In contrast, in non-dormant seeds imbibed for 24 h, treatment with 0.8 M KCl reduced the height of 80S peak (Supplemental Figure 1C) thus suggesting that a part of monosomes were RNA free. The increase of the 80S peak in non-dormant seeds therefore did not correspond to active ribosomes bound to mRNA.
Regulation of polysome loading by mRNA sequence features and by translational efficiency
The polysomal ratio (proportion of individual mRNA species in polysomes, P/T, Figure 3A) was determined for transcripts detected in polysomal fractions. While the distribution of polysomal ratios was similar for both durations of imbibition, it appeared significantly (p<0.0001) different between dormant and non-dormant seeds (Figure 3A). This distribution shows that the transcripts which were more abundant in dormant seeds presented a lower polysomal ratio (i.e. a higher number of transcripts with a polysomal ratio lower than 80 %) than the transcripts more abundant in nondormant seeds (a higher number of transcripts with a polysomal ratio of 92 %), whatever the duration of imbibition (Figure 3A). Major characteristics of 5’UTR and 3’UTR (length, GC content) of differentially abundant transcripts in translatome with a described 5’UTR described in TAIR database (Supplemental Table S2) have been analyzed in relation with their polysomal ratio. At both duration of imbibition, higher values of polysome ratios were observed when 5’UTR length was between 50- 80 nucleotides (nt) and P/T was lower for mRNAs with short (<25 nt) or long (> 100 nt) 5’UTR lengths, whatever seeds were dormant or not (Figure 3B). However, the relationship between P/T and 5’UTR length was not affected by dormancy since the slopes of the curves shown in Figure 3B were similar.
The relationship between polysome loading and GC content in 5’UTR revealed differences between
dormant and non-dormant seeds (Figure 3C). Low GC contents (20-30 %) in 5’UTR were associated with high polysomal ratio and higher GC content significantly decreased ribosome loading (Figure 3C). The effect of GC content on P/T was considerably more pronounced in dormant than in nondormant seeds since P/T decreased to 25 % in dormant seeds but only to 55 % in non-dormant ones when GC content increased to 60 % (Figure 3C). The effect of 3’UTR features on polysomal ratio was also investigated but did not allow to evidence any relationship between length or GC content and the ability of seeds to germinate (Supplemental Figure S3). Over-represented motifs in 5’UTR of polysomal transcripts more abundant (analysis on 100 transcripts) in dormant and non-dormant seeds were determined with the unsupervised multiple expectation maximization for motif elicitation (MEME) algorithm (Bailey et al., 2009) (Figure 3D). We identified U-rich motifs in the 5’UTR of polysomal RNAs from dormant seeds and the same consensus motif “AGAGAGAAGA” at both durations of imbibition in polysomal transcripts from non-dormant seeds (Figure 3D). In all cases, the values of motif enrichment (ME, which corresponds to the factor of enrichment of this motif in comparison with its occurrence in the whole Arabidopsis genome) and of E-value (which refers to its statistical significance) demonstrated the high specificity of the motifs (Figure 3D). Putative uORFs were searched in 5’UTR of all the transcripts associated with polysomes (listed in Supplemental Dataset S4). More than 75 % of dormant seeds displayed few uORFs (1-3), whereas transcripts with more than 3 uORFs were more frequent in dormant seeds (Table I). Figure 3E shows that the polysomal ratio decreased with the increasing number of uORF per transcript but this effect was more pronounced in dormant seeds. The longer the length of uORF the lower the the transcripts did not present any uORF (Table I) but transcripts from non- polysomal ratio was (Figure 3F); however, the inhibition of polysome loading by the length of uORF appeared to be stronger for transcripts more abundant in non-dormant seeds (higher slope values on the graph).
Table of contents :
I. State of the art
I.1 Seed dormancy and germination
I.1.1 Arabidopsis seed development and structure
I.1.2 Seed germination
I.1.3 External factors regulating germination
I.1.4 Seed dormancy
Factors of dormancy
I.1.5 Hormonal regulation of dormancy and germination
ABA and GA balance
I.2 Transcriptional regulation of gene expression
I.2.1 Transcriptional regulation in plants
I.3 Post-transcriptional regulation of gene expression
I.3.1 Regulation of mRNA translation in plants
Regulation of translation initiation
Regulation of eIF2 activity
Regulation of eIF4E and eIFisoE activity
5′ to 3′ Exoribonucleases
Specialized decay pathways
I.4. Objectives of the thesis
II. Fluctuation of Arabidopsis seed dormancy with relative humidity and temperature during dry storage
II.2 Material and methods
III. Germination potential of dormant and non-dormant Arabidopsis seeds is driven by distinct recruitment of mRNAs to polysomes
III.4 Materiels and methodes
IV. 5′ to 3′ mRNA decay contributes to the regulation of Arabidopsis seed germination by dormancy