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JXB Advance Access originally published online on December 14, 2006
Journal of Experimental Botany 2007 58(3):425-437; doi:10.1093/jxb/erl211
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Identification of transcripts potentially involved in barley seed germination and dormancy using cDNA-AFLP

Juliette Leymarie*, Elisabeth Bruneaux, Stéphanie Gibot-Leclerc and Françoise Corbineau

Université Pierre et Marie Curie-Paris 6, EA2388 Physiologie des semences, Site d'Ivry, Boîte 152, 4 place Jussieu, F-75005 Paris, France

* To whom correspondence should be addressed. E-mail: juliette.leymarie{at}upmc.fr

Received 18 July 2006; Revised 1 September 2006 Accepted 21 September 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Freshly harvested barley seeds are considered as dormant since they do not germinate at temperatures above 20 °C. This dormancy is broken during dry storage. Molecular regulation of dormancy was investigated using cDNA-AFLP to identify transcripts differentially expressed in dormant and non-dormant embryos. Transcript patterns in embryos from dry dormant and non-dormant seeds and from both seeds imbibed for 5 h at 30 °C, a temperature at which dormancy is expressed, were compared. Thirty-nine Transcript-Derived Fragments (TDF) that were reproducibly differentially expressed among treatments were identified, and 25 of these were cloned and sequenced. Among these, eight transcripts were observed to be differentially expressed during after-ripening, seven of which decline, probably due to post-maturation degradation. HV13B, TDF identified as having homology to fructose-6-phosphate-2-kinase/fructose-2,6-biphosphatase, may have a role in the maintenance of dormancy in barley and probably in other cereals. During the first 5 h of imbibition, there was expression of 24 TDF which was apparently independent of dormancy, revealing putative epigenetic regulation. This was typified by HV44A, a SET domain protein. Seven TDF differentially expressed, and especially HV12D, HV42B, and HV32B, in dormant and non-dormant seeds were potential signalling elements. HV12D had homology with an ARIADNE gene which could be implicated in ABA signalling.

Key words: After-ripening, barley, cDNA-AFLP, gene expression, germination, seed dormancy


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Dormancy of barley seeds, as in other cereals from temperate climates, is typified by an inability of freshly harvested seeds to germinate at temperatures higher than 20 °C, while they germinate readily at relatively low temperatures (10–20 °C) (Lenoir et al., 1986; Corbineau and Côme, 1996). This dormancy can be regarded as a relative phenomenon, the expression of which depends on the incubation temperature. In barley, it is mainly due to the glumellae, which fix oxygen through the oxidation of phenolic compounds, limiting oxygen supply to the embryo (Corbineau and Côme, 1980; Lenoir et al., 1986). In addition, hypoxia imposed by the glumellae interferes with abscisic acid (ABA) metabolism in the embryo and increases the sensitivity of the embryo to ABA (Benech-Arnold et al., 2006). Although embryos isolated from dormant seeds are able to germinate at high temperatures, they are more sensitive to the environmental factors (temperature, oxygen, water potential of the medium) than those from non-dormant seeds (Corbineau and Côme, 1996).

ABA has been invoked to be involved in both the imposition and the maintenance of seed dormancy (McCarty, 1995; Bewley, 1997). Indeed, analysis of ABA deficient or -insensitive mutants of various species that display low dormancy or pre-harvest sprouting, has provided strong evidence that ABA is clearly implicated in the onset of dormancy during seed development (McCarty, 1995; Bewley, 1997). Dormancy of barley grains also appears to be under ABA control (Wang et al., 1998; Benech-Arnold et al., 2006). Gibberellins can overcome dormancy in cereals and appear not to be directly involved in the control of dormancy, but rather are important in the promotion of germination, acting downstream from ABA (Bewley, 1997).

Dormancy in cereal seeds is gradually eliminated during dry storage. However, the mechanisms of this phenomenon, called after-ripening, in particular the reorientation of the genetic programme associated with the transition from a dormant to a non-dormant state, are currently not known. Apparent local gene expression during after-ripening was reported in Nicotiana tabacum (Leubner-Metzger, 2005) and Nicotiana plumbaginifolia (Bove et al., 2005), however, transcription of genes at the low moisture contents at which after-ripening can proceed is debatable (Vertucci and Farrant, 1995).

Numerous studies using mRNA differential screening, microarrays or proteomics have characterized transcripts or proteins, the expression of which during imbibition correlates with dormancy or germination (Ried and Walker-Simmons, 1990; Goldmark et al., 1992; Johnson et al., 1995; Stacy et al., 1996; Gallardo et al., 2001, 2002; Potokina et al., 2002; Watson and Henry, 2005; Toorop et al., 2005). Many of the genes identified are involved in desiccation tolerance, stress responses, DNA repair, and various aspects of germination completion, but only few of them might be specifically involved in dormancy maintenance or release (Bove et al., 2005; Gubler et al., 2005).

In cereals, several ABA-responsive mRNAs persist in embryos during imbibition of dormant seeds, but decline during germination of non-dormant ones (Ried and Walker-Simmons, 1990; Morris et al., 1991; Li and Foley, 1995). In barley for example, HVA1, coding for a late embryo abundant (LEA) protein, is expressed in embryos from both dormant and non-dormant seeds, but HVA1 mRNA level declines faster upon imbibition in non-dormant seeds than in dormant ones (Hong et al., 1992). Differential screening of a dormant wild oat library allowed Ranford et al. (2002) to identify a barley cDNA PM19 encoding a putative plasma membrane protein. The PM19 transcripts are expressed during late embryogenesis and disappear during germination of non-dormant seeds while the mRNA levels remain high in dormant embryos. In barley, peroxiredoxin transcript PER1 is accumulated in developing dormant grains while it disappears during germination (Stacy et al., 1996, 1999). A similar transcript pattern is observed in Arabidopsis and is regulated through ABI3 (Haselkas et al., 1998, 2003). Goldmark et al. (1992), using differential screening for mRNAs in Bromus secalinas, identified pBS128 transcript potentially involved in dormancy. Although its orthologue was identified in barley (Aalen et al., 1994), no putative function was assigned. Johnson et al. (1995), using differential display, identified in wild oat five clones, two dormant-specific and two non-dormant specific, but only one (AFN3) had homology with a previously described sequence (glutathione peroxidase).

Genetic approaches using QTL analyses have also been performed in order to detect genes involved in seed dormancy. Fennimore et al. (1999) have constructed a dormancy model for wild oat, suggesting the involvement of three loci, but those authors have not been able to characterize a candidate gene specific to dormancy release in this species. In wheat, several genes or chromosomal regions affecting dormancy have been identified (Warner et al., 2000; Kato et al., 2001; Himi et al., 2002; Miura et al., 2002; Noda et al., 2002; Osa et al., 2003). Four and five QTLs were revealed to be associated with dormancy in Arabidopsis (Alonso-Blanco et al., 2003) and rice (Wan et al., 2006), respectively.

Among genome-wide expression analyses, DNA microarrays is the most powerful tool for organisms for which the complete genome sequence is known, or for which large cDNA collections are available. In barley, the GeneChip has only recently been made available (Close et al., 2004) and studies using cDNA arrays to reveal gene expression on grains have only recently been reported (Potokina et al., 2002; Watson and Henry, 2005; Druka et al., 2006; Sreenivasulu et al., 2006). For most cultivated plant species, as is the case for barley, the cDNA-AFLP technique provides a more appropriate tool for global gene expression analyses (Breyne and Zabeau, 2001). This technique, derived from the differential display technique, uses restriction enzymes to generate cDNA specific tags which, followed by PCR amplification, allows the identification of transcripts specifically expressed under certain physiological conditions in various systems (Bachem et al., 1996; Durrant et al., 2000) including seed germination in bean (Aubry et al., 2003) and Arabidopsis (de Diego et al., 2006), and in seed development in flax (Gutierrez et al., 2006).

The aim of the present work was to characterize, by cDNA-AFLP, changes in transcript expression in embryos of barley seeds in relation to dormancy independently of any growth process. Transcript expression patterns were studied in embryos isolated from dormant and non-dormant seeds before imbibition (dry seeds) and after 5 h of imbibition at 30 °C, a temperature at which dormancy is expressed and a duration of imbibition which does not result in early cellular changes associated with further radicle elongation. Transcripts involved in post-maturation, seed imbibition, and dormancy were identified, and their putative functions are discussed.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Conclusion
 References
 
Plant material and storage
Barley (Hordeum vulgare L., cv. Pewter) seeds, harvested in July 2002 and 2004, and kindly provided by the ‘Coopérative de Toury’ (Eure et Loir, France), were used throughout this study. Experiments were carried out with dormant grains, which were stored at –20 °C from harvest until the experiments began in order to maintain their initial dormancy (Lenoir et al., 1983), and non-dormant grains, which were stored dry in the open air for 2 months at 25 °C in order to break their dormancy (Corbineau and Côme, 1996). After breaking of dormancy, non-dormant seeds were also placed at –20 °C until experiments started.

Germination assays
Germination assays were performed by placing whole grains (50 grains per dish, three replicates) in Petri dishes on a layer of cotton wool imbibed with deionized water. Assays were carried out at temperatures ranging from 5 °C to 35 °C. A grain was regarded as having germinated when the radicle protruded through the seed covering structures (seed coat+pericarp and glumellae). Germination counts were made every day for 7 d.

RNA extraction
Embryos were isolated from the endosperm using a sharp scalpel blade, immediately frozen in liquid nitrogen, and then stored at –80 °C. For each extract, 30 embryos were ground in liquid N2, and total RNA was extracted by a hot phenol procedure according to Verwoerd et al. (1989). RNA concentration was determined spectrophotometrically at 260 nm.

cDNA-AFLP
A cDNA-AFLP method was adapted from Bachem et al. (1996, 1998). Poly(A) RNA was purified from total RNA using the GeneElute kit (Sigma, St Louis, USA) according to the manufacturer's instructions. Efficiency of the purification was checked by agarose gel electrophoresis and the concentration was determined spectrophotometrically at 260 nm.

After DNase-1 treatment (Sigma) the cDNA first strand was synthesized using a mix of dT oligonucleotides (T(18), T(17)A, T(17)C, T(17)G) and Revertaid H minus M-MuLV reverse transcriptase (Fermentas, Burlington, Canada) (2 h at 42 °C). The second strand was synthesized at 16 °C for 2 h with DNA-polI and ligase in presence of RNase H (Fermentas). The resulted double-stranded cDNA was phenol-extracted, ethanol-precipitated, and resuspended in 30 µl of water. Half of this volume was checked using agarose gel electrophoresis in order to observe an expected smear between 100 bp and 3000 bp. The rest of cDNA was subjected to AFLP template production (Vos et al., 1995) using the restriction enzymes Mse1 (Tru1l, Fermentas; 2 h at 65 °C) and EcoR1 (Fermentas; 2 h at 37 °C). EcoAd (5 µM) and MseAd (50 µM) adaptors were annealed in TRIS buffer (250 mM pH 7.5, MgCl2 5 mM) from oligonucleotides Eco-ad1, Eco-ad2 and Mse-ad1, Mse-ad2, respectively. Adaptors were ligated with T4-DNA ligase (Fermentas) for 3 h at 37 °C and 1/10 of this reaction volume was used for preamplification with Mse-P and Eco-P primers in 25 µl reaction volume. Preamplification was initiated at 94 °C for 1 min and at 72 °C for 30 s followed by 25 cycles at 94 °C for 30 s, 51 °C for 30 s, 72 °C for 1 min 30 s, and terminated at 72 °C for 5 min. The products of the preamplification were checked by agarose gel electrophoresis (expected smear between 100 bp to 1000 bp) and their concentrations were determined spectrophotometrically at 260 nm. They were diluted to obtain a final concentration of 10 ng µl–1. The AFLP reactions were made with 5 µl of diluted preamplified solutions, MseX (50 ng) and EcoY (10 ng) primers. After initial denaturation (94 °C for 1 min), 11 cycles were performed with touchdown annealing (94 °C for 30 s, 62 °C to 52 °C in 0.7 °C steps for 30 s, 72 °C for 1 min) followed by 25 cycles (94 °C for 30 s, 52 °C for 30 s, 72 °C for 1 min) and final elongation (72 °C for 5 min). For PCR, 68 combinations of two base extensions (denoted as NN) were used.

All oligonucleotides were obtained from Eurogentec (Seraing, Belgium) and amplifications were performed using a personnal mastercycler (Eppendorf, Hamburg, Germany). Oligonucleotide sequences are given below:

Eco-ad1: CTCGTAGACTGCGTACC
Eco-ad2: AATTGGTACGCAGTCTAC
Mse-ad1: GACGATGAGTCCTGAG
Mse-ad2: TACTCAGGACTCAT
EcoP: GTAGACTGCGTACCAATTC
MseP: GACGATGAGTCCTGAGTAA
EcoX: GACTGCGTACCAATTCNN
MseY: GATGAGTCCTGAGTAANN

Polyacrylamide gel electrophoresis
Fragments were separated on a sequencing polyacrylamide gel (6% bis-acrylamide, 7 M urea, 1x TBE) using Sequigen system (Bio-Rad, Hercules, CA, USA). The glass plates were treated by Plus One Repel-Silane (Amersham Biosciences, Little Chalfont, UK) and {gamma}-methacryloxypropyl-trimethoxysilane (Sigma) following the manufacturer's instructions. After 4 h of migration (2 kV, 50 °C) in 1x TBE buffer, the fragments were visualized by silver staining according to Bassam et al. (1991).

Isolation and sequencing of the transcript-derived fragments (TDF)
Fragments were eluted from silver-stained gels using the procedure of Frost and Guggenheim (1999) with modifications. The band of interest was rehydrated for 5 min with 2x PCR buffer (20 mM TRIS–HCl pH 8.8; 100 mM KCl; 0.16% Tween 20), excised from the gel, and transferred in 100 µl 2x PCR buffer for 10 min at room temperature. The band was then transferred to new 2x PCR buffer (100 µl) and heated at 94 °C for 90 min. Amplifications were performed with appropriate primers with 5 µl of this solution 10 times diluted. PCR reactions were initiated at 94 °C for 1 min followed by 35 cycles at 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min, and terminated at 72 °C for 10 min.

Cloning was performed from fresh PCR products with the pCR®2.1-TOPO® TA cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and using chemical transformation of one shot® DH5{alpha}TM competent cells using kanamycin as the selecting agent. After plasmid purification using a miniprep Plasmix kit (Talent, Trieste, Italy), the insert size was checked by PCR amplification using M13 primers and the insert sequenced by Genomexpress (Meylan, France). Similarity studies were performed with BLAST program (Altschul et al., 1997) using the NCBI website (www.ncbi.nlm.nih.gov/BLAST/) and the TIGR Barley Gene Index database (Quackenbush et al., 2001; www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=barley).

Real-time quantitative RT-PCR
Total RNA was treated with DNase I (Sigma) and then was reverse transcribed with Revertaid H minus M-MuLV RT (Fermentas). After enzyme inactivation, the first strand cDNA obtained was checked by agarose gel electrophoresis. The amplifications were performed with real-time PCR (iCycler iQ, Bio-Rad) using 5 µl of x50 diluted cDNA solution. The primers used are listed in Table 1. As an internal standard, a fragment of barley actin gene (GI24496451) was used. Real time PCR reactions were performed with the Absolute qPCR Syber Green Fluorescein mix (Abgene, Epsom, UK) and 0.25 µM of each primer in a 25 µl reaction volume. They were initiated at 94 °C for 15 min followed by 40 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s. Calculations of Critical threshold (Ct) and relative expressions were performed using the iCycler iQ software (Bio-Rad).


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  		Table 1. Primers used for each TDF and actin gene (internal control) for real-time PCR amplifications

 
Northern blots
Total RNA was separated (10 µg per lane) in 1% agarose-formaldehyde gel (Sambrook and Russel, 2001). RNA loading was checked by ethidium bromide staining. RNA was transferred to nylon filter (Biodyne B, Pall, New York, USA) by capillary action with 10x SSC (Sambrook and Russel, 2001) and fixed by UV crosslinking (Stratalinker, Stratagene, La Jolla, CA, USA).

DNA probes were labelled with {alpha}32P-dCTP (Amersham Biosciences) using ‘Ready-To-Go DNA labelling beads’ kit (Amersham Biosciences) and purified with the ‘ProbeQuant G-50 Micro Columns’ (Amersham Biosciences) as described by the manufacturer. The PCR products obtained after M13 amplification were used as probes for northern blot analysis. Membranes were hybridized at 65 °C in 0.5 M sodium phosphate buffer (pH 7.2), 5% SDS, and 10 mM EDTA. A first wash was performed at 65 °C in 1x SSC, 0.1% SDS and a second wash at 65 °C in 0.1x SSC, 0.1% SDS. The membranes were analysed with a Phosphorimager (Storm 840, Amersham Biosciences) and ImageQuant software (Amersham Biosciences) according to the manufacturer's instructions.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Conclusion
 References
 
Physiological model
Figure 1 shows the effects of temperature on the germination percentage obtained after 7 d from dormant (i.e. freshly harvested seeds) and non-dormant seeds (i.e. seeds stored dry for 2 months at 25 °C). Although freshly harvested seeds do germinate at permissive temperatures of up to 20 °C, these seeds were consider ‘dormant’, since germination is virtually precluded above 20 °C. Almost 90–100% of the seed population germinated within 7 d at 10–20 °C, whereas only 10–15% germinated at 30 °C. The insert in Fig. 1 shows that germination of almost all of the non-dormant seed population occurred within 24 h at 30 °C, and that the lag time for visible germination of these non-dormant seeds was around 12–13 h. However, the radicle of some embryos had already protruded from the seed coat+pericarp under the glumellae after 8 h. It is assumed then that 5 h of imbibition resulted in the realization of the germination sensu stricto in non-dormant seeds, whereas this phenomenon was not possible in dormant seeds, without inducing molecular and cellular events associated with subsequent radicle growth.


Figure 1
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  		Fig. 1. Effects of temperature on the germination percentages obtained after 7 d with dormant (circles) and non-dormant (squares) barley seeds. Means of three measurements. The vertical bar indicates the largest SD. Insert: time-course of germination at 30 °C of dormant and non-dormant seeds during the first 24 h of imbibition.

 
Expression patterns
Gene expression cDNA-AFLP analyses were performed in embryos isolated from dormant and non-dormant seeds before (dry state) and after 5 h of imbibition in water at 30 °C, the temperature at which dormancy is expressed. With the 68 primer combinations tested, 39 reproducible (i.e. same expression profile in seeds harvested in 2002 and 2004) transcript-derived fragments (TDF) were identified (Table 2). This comparison of expression changes between two seed batches resulted in discarding half of the transcripts that were detected after a first analysis. Moreover, when expression was not confirmed by further analyses (northern blots or RT-PCR) TDF have not been mentioned here (HV33D, HV42A, HV45D, HV62B, HV62D, HV67C, HV67D).


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  		Table 2. cDNA-AFLP expression pattern of transcript-derived fragments (TDF) identified as polymorphic in embryos of dormant (D) and non-dormant (ND) seeds before (dry state) and after 5 h of imbibition in water at 30 °C

 
The TDF identified can be classified in three groups (I, II, and III) and various sub-groups (a–d) according to their expression profiles (Table 2). Eight TDF (group I) corresponded to changes occurring in dry embryos during after-ripening. Among them, seven TDF disappeared during dry storage (expression patterns Ia, Ib, and Ic), whereas one TDF increased in abundance (expression pattern Id). Two TDF from group I also showed differences in expression during imbibition. The more significant group (II) consisted of 24 TDF, among which the expression of 16 (IIa) was induced and eight (IIb) disappeared during imbibition in both dormant and non-dormant seeds, i.e. independently of dormancy. The third group (expression patterns IIIa and IIIb) consisted of seven TDF, the expression of which was also related to the imbibition process, but with a different pattern in dormant and non-dormant seeds.

Identification of TDF
Among the TDF identified as polymorphic, 25 reproducible TDF have been cloned and sequenced. Since two fragments were redundant and seven expression patterns were not confirmed (mainly after-ripening changes), 16 sequences are shown in Table 3.


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  		Table 3. Transcript-derived fragments (TDF) identified as polymorphic by cDNA-AFLP and sequenced

 
Similarity determination was performed using the BLAST program (Altschul et al., 1997) against the GenBank and EMBL database (nucleotide, EST, and protein) and TIGR database. BLAST Expect Value (E) is reported. Similarity with barley sequences generally corresponded to EST or Tentative Consensus Sequence derived from several EST (accession number beginning with TC according TIGR nomenclature), because public genomic data concerning barley are scarce. In most cases (12/16), a known barley expressed sequence corresponded to the TDF sequenced with more than 98% identity (Table 3). Comparison with genes from other plant species allowed identification of eight TDF showing high level of similarity with genes with known or putative functions (Table 3). Tables 3 and 4 show that changes in transcript expression that occurred during imbibition, independently of dormancy (group II), are related to signalling (HV44A), metabolism (HV41A), or detoxification (HV13A). Alterations related to dormancy (groups I and III) are related to diverse functions such as signalling (HV12D, HV42B and HV32B), metabolism (HV13B, and HV47A), DNA replication (HV45E), and detoxification (HV75A).


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  		Table 4. Classification of the identified TDF according to their putative functions and their expression patterns as defined in Table 3

 
Validation of differential expression revealed by cDNA-AFLP
After identification by cDNA-AFLP, gene expression was further studied for 15 TDF, using northern blot or RT-PCR, since, in many instances, the northern blot signal was too low. In most cases the expression pattern was confirmed (indicated in Table 3) but seven TDF (five being identified as altered during after-ripening) presented no significant variation using RT-PCR or northern blot. As an illustration, Figs 2 and 3 show northern and RT-PCR confirmation of selected TDF, respectively.


Figure 2
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  		Fig. 2. HV41A (S-adenosyl methionine decarboxylase) (A) and HV32B (B) transcript expression in embryo isolated from dry seeds or seeds imbibed for 5 h at 30 °C in dormant (D) or non-dormant state (ND). Data from northern blot (mean of two replicates±arithmetical spread) were expressed in arbitrary units.

 

Figure 3
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  		Fig. 3. HV13A (A) and HV75A (B) transcript expression in embryo isolated from dry seeds or seeds imbibed for 5 h at 30 °C in dormant (D) or non-dormant (ND) state. Data from real time RT-PCR (mean of six replicates ±SE) were expressed in arbitrary units.

 
HV41A was present in dry seeds (Table 3), but its expression decreased upon imbibition. Northern blot analyses confirmed this expression profile, with two times less expression after 5 h of imbibition (Fig. 2A). Similarly, northern blot and RT-PCR analyses confirmed the cDNA-AFLP expression profile of HV32B (Fig. 2B); its expression was low in dry seeds and imbibed non-dormant one, but was 2-fold up-regulated in dormant imbibed seeds. HV13A (Fig. 3A) was clearly induced by imbibition in both types of seeds while HV75A was only induced in non-dormant imbibed ones (Fig. 3B).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Conclusion
 References
 
Validity of the system
Using the cDNA-AFLP technique, it was possible to detect an alteration in gene expression during after-ripening and during the initial stages of germination in barley seeds. Half of TDF identified had to be disregarded for further analysis due to an inability reliably to confirm their differences among samples. This might be due to technical reasons, the cDNA-AFLP technique not being sensitive enough to detect slight expression changes, but it is also highly possible that significant gene changes are not detected due to biological heterogeneity among distinct seed samples, since barley seeds were not grown in controlled conditions and were harvested in 2002 and 2004. Among about 100 TDF visualized for each primer couple, around 1% of cDNA fragments can be estimated as being differentially expressed in the four conditions studied. In Nicotiana plumbaginifolia, seeds placed under eight germination conditions allowed Bove et al. (2005) to identify 6.8% of differentially expressed genes among 15 000 TDF analysed. De Diego et al. (2006) identified 35 TDF after 64 combination analysis and comparison between dry and 24 h-germinated seeds of Arabidopsis thaliana. Using microarrays, among the 12 500 RNAs detected in Arabidopsis seeds, the expression of more than 10 000 remained unchanged during seed germination, suggesting that around 20% of gene expression are altered (Nakabayashi et al., 2005). In barley, Watson and Henry (2005), using cDNA arrays, have identified around 10% of the cDNA spotted as differentially expressed during germination. Thus, these results indicate relatively less alteration in gene expression than in other studies, but in the current work, germination was carried out for only 5 h at 30 °C, i.e. before radicle protusion had occurred.

The confirmation of expression by RT-PCR or northern blot was not performed for all transcripts. While in most cases, one or other of the techniques allowed confirmation, in some instances neither technique was conclusive. In a recent paper, van Raemdonk et al. (2005) reported confirmation by RT-PCR for 19/25 TDF detected, and that reverse northern analysis on 106 TDF did not confirm cDNA-AFLP in most cases. This was suggested to be due to hybridization of homologous cDNA or that the signals were too low. In our experiments, most of the false positives corresponded to after-ripening changes that were not reproducible. A high biological variability in barley grains could be responsible for these artefacts.

Although genomic data are not available for barley, the EST databases allowed us to identify for most cases (17/23) an homology with a barley expressed sequence. However, unfortunately, a putative function of the gene could not always be associated with the sequence. Watson and Henry (2005) sequenced around 80 cDNAs from barley expressed during germination and found homology with barley or wheat for all with 64% presenting homology with proteins having a putative function.

Changes in gene expression during after-ripening
Stored mRNA in mature dry seeds is universal in plant species (Comai and Harada, 1990; Bewley, 1997; Nakabayashi et al., 2005) and is thought to play a role in protein synthesis for both late embryo development and for the early stages of germination. The fact that 7/8 of the variations reported here corresponded to a decrease in expression during dry storage is thus not surprising. There are indications that a global decrease of RNA occurs to some extent during dry storage, due to RNA degradation (Priestley, 1986). This lost RNA may code for polypeptides that are important during seed maturation, but less relevant in the regulation of dormancy. In wild oat, the expression of AV1 and Z1 mRNA decreased during after-ripening and was more important in the dormant genotype (Johnson and Dyer, 2000). But the lack of correlation between mRNA levels and the dormancy status of individual grains indicated that there was no causal relationship between these genes' expression and dormancy. Those authors proposed that ageing, rather than a specific degradation, was responsible for this pattern of RNA availability. In barley, HV33C was lost during after-ripening and did not survive imbibition (Table 3), suggesting that it may code for proteins which are essential during seed development, but not for germination. Their disappearance during imbibition probably resulted from degradation during water uptake (Priestley, 1986). HV45A, HV45B, HV45E mRNA were also lost during after-ripening, but they are expressed in embryos of both dormant and non-dormant seeds within 5 h of imbibition (Table 3). HV45E has homology with putative rice DNA polymerase V. In E. coli, the DNA polymerase V participates in SOS response, which allows the cell to cope with UV- and chemical-induced DNA damage. Polymerase V catalyses translesion synthesis by replacing a replicative polymerase III that stalls when encountering a damaged template base. Translesion synthesis results in mutations targeted to the sites of DNA damage that leads to dramatic increase in mutagenesis (Schlacher et al., 2006). Its expression during the early stages of germination suggested a possible role in DNA repair, but its reduction during after-ripening is difficult to explain.

It is assumed that neither transcription nor translation occurs at water contents lower than 25% dry weight basis (Vertucci and Farrant, 1995). Dyer (1993) did not detect any changes in translation products from dormant and non-dormant Avena fatua embryos in a dry state. By contrast, Bove et al. (2005) and Leubner-Metzger (2005) recently reported that gene transcription and translation were possible in dry seeds of Nicotiana plumbaginifolia and N. tabacum. In barley, one TDF (HV13B) is induced during after-ripening and persists during imbibition of non-dormant seeds (Table 3). These results suggest that more hydrated areas within the tissues or the cells may exist to allow such a gene expression. However, in spite of the reproducible results, it cannot be excluded that this apparent transcription was due to changes in extractability or partial degradation of RNAs. HV13B has homology to fructose-6-phosphate-2-kinase/fructose-2,6-biphosphatase. Despite repeated northern blots, no signal could be detected, suggesting low expression. While real-time RT-PCR analyses confirmed the AFLP profile, the differences observed between the treatments were weak (increase of 50% during after-ripening; data not shown), and it is difficult to conclude whether this putative gene product has a function in the breaking of dormancy during after-ripening of barley seeds. The enzyme has been shown to act as a kinase and as a phosphatase depending on the cellular metabolism. It is thus difficult to explain the consequence of changes in this transcript, although fructose-2,6-bisphosphate (Fru-2,6-P2) may play a role in the dormancy processes since it reaches transiently high values in embryos of non-dormant oat seeds and of dormant red rice and oat seeds placed in conditions that break dormancy (Larondelle et al., 1987; Footitt and Cohn, 1995).

Changes in gene expression during imbibition
Dyer (1993) and Nakabayashi et al. (2005) have demonstrated that 6 h imbibition resulted in differential gene expression in various species, which depended on the stage of dormancy. In the current study, after 5 h of imbibition, the expression of 24 TDF were altered independently of dormancy while seven TDF were altered differentially in dormant and non-dormant seeds.

Sixteen transcripts absent in dry seeds accumulated during imbibition (Tables 2, 3, expression profile IIa). Among them, the HV13A sequence showed high similarity with Protein Disulphide Isomerase (PDI), which is an essential protein that catalyses disulphide bond formation (Freedman et al., 1994). In wheat, PDI is more highly expressed in 3-d-old germinating seedlings than in dry seeds and its expression is higher in the aleurone layer than in embryos (Livesley et al., 1992). Three PDI sequences have already been identified in barley (Chen and Hayes, 1994), but they presented no homology with the HV13A sequence. Our results suggest then that there are either four genes encoding PDI in barley, or HV13A could belong to PDI-like family (Freedman et al., 1994) and have a different function than PDI. A potential role in detoxification is possible due to the presence of a thioredoxin domain. HV44A was homologous to barley TC145904 and to a maize SET domain protein SDG117 (Springer et al., 2003). SET domain proteins are protein lysine methyltransferase enzymes, widely represented in eukaryotic genomes. They have been implicated in epigenetic mechanisms of gene expression by methylation of lysine residues in histone and other proteins (Alvarez-Venegas and Avramova, 2000). Demethylation during imbibition might be involved in the recovery of metabolic activity.

Several transcripts are known to disappear during imbibition. Classical examples are LEA proteins that accumulate during the acquisition of desiccation tolerance and decline during germination (Ried and Walker-Simmons, 1990; Hong et al., 1992). In barley, eight TDF disappeared during imbibition. Among them HV41A TDF (Fig. 2A) corresponds to the orthologue of the rice S-adenosyl methionine decarboxylase (AdoMetDC; Table 3). Further expression analysis of this gene showed that this effect of imbibition is also present when dormancy is not expressed at 20 °C, but there is induction of its expression after 24 h imbibition (data not shown). Besides being the source for methylation in plant cells, AdoMet is either decarboxylated, allowing the synthesis of polyamines (spermine and spermidine), or it is the precursor ethylene. It has been shown in chick pea that these two pathways might compete during germination (Gallardo et al., 1995). The presence of HV41A TDF in dry mature seeds is in agreement with results obtained by Radchuk et al. (2005) who demonstrated that the AdoMetDC transcript is induced during the maturation drying phase of seed development in barley. Polyamines are known to be involved in a wide range of cellular physiological processes including chromatin organization, mRNA translation, cell proliferation, apoptosis, and stress responses (Bouchereau et al., 1999). HV41 sequence is more similar to the rice AdoMetDC2 than to rice AdoMetDC1. AdoMetDC1 is ubiquitous and abundant compared with AdoMetDC2, which might probably respond more to environmental signals (Franceschetti et al., 2001). Other studies have shown the importance of methionine metabolism in Arabidopsis and Medicago truncatula seed germination (Gallardo et al., 2001, 2002, 2003).

In barley, five TDF were induced specifically in imbibed non-dormant seeds, while two TDF were specific to dormant ones. HV12D, expressed only in non-dormant seeds, was homologous to a barley RNA (gi:9435588, EST BE437746 [GenBank] ) and to several wheat EST from various tissue types (including leaves, grain and pistils). Its sequence contained a zinc finger domain (ZnF-RBP) and showed homology to a putative rice ARIADNE gene. The ARIADNE protein family, recently identified in plants (Mladek et al., 2003), is characterized by a RING-finger that interacts with the ubiquitin-conjugation E2 enzyme in animals (Aguilera et al., 2000). Its expression during the early stage of imbibition suggests that the ubiquitin pathway might be involved in germination. Moreover, the ubiquitin/26S proteasome pathway has been already shown to be implicated in ABA signalling and particularly in seed germination (Smalle et al., 2003). HV47A is a putative galactosyltransferase, an enzyme transferring a galactose residue to polysaccharides, lipids or proteins. The induction of such an enzyme could be related to the resumption of metabolism during germination. However, the homology with a gene coding a galactosyltransferase implicated in plant cell wall matrix is not significant (Edwards et al., 1999). A recent BLAST analysis of the AFN1 sequence, a transcript identified by Johnson et al. (1995) to be specific to non-dormant wild oat, shows homology to a putative glucosyltransferase. HV42B showed similarity with a rice sequence annotated as putative Cf2/Cf5 disease resistance protein, but this function has not yet been verified. The homology is due to the Leucine Rich Repeat domain (LRR) which is present in protein families other than Cf2/Cf5, suggesting a ligand-binding function potentially involved in other signals than a response to just biotic stress (Napier, 2004). HV75A corresponded to TC146301 of barley, annotated as similar to a thioredoxin homologue from Arabidopsis, and, thus, being potentially involved in detoxification mechanisms. These data indicated that thioredoxin might play a role in dormancy, which is in agreement with Côme et al. (1988) who suggested that the reduction of proteins by a thioredoxin type may activate enzymes necessary for germination.

Two sequenced TDF (HV32B and HV65A) were specific to imbibed dormant seeds. HV65A is identical to a barley transcript already detected in the germinating shoot, but with no putative function. The HV32B sequence presented a PDZ domain of trypsin-like serine protease. Trypsin-like serine protease might be implicated in proteolysis occurring during germination (Tsuji et al., 2004). With the expression that occurs only in dormant seeds, it is the similarity of the PDZ domain that appears more important than the complete protease. PDZ domain is a protein–protein recognition module found in diverse signalling processes. Several PDZ-domain-containing proteins play an important role in transport, localization, and assembly of signalling complexes in animals (Harris and Lim, 2001). In plants, the ZKT protein contains a PDZ domain and may act as a molecular adaptator regulated by phosphorylation in wound responses (Ishikawa et al., 2005). In dormant imbibed barley seeds, specific signalling cascades may be activated in order to block the germination process, and then HV32B could be implicated in such regulation.


   Conclusion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
References
 
In summary, the cDNA-AFLP technique was successfully used to identify genes that are differentially expressed in the dry state or after 5 h of imbibition in embryos from dormant and non-dormant barley seeds. The reduction of expression of seven genes during after-ripening might be more related to a global decrease of RNA during storage than a real regulation of dormancy. The regulation of expression of the fructose-6-phosphate-2-kinase/fructose-2,6-biphosphatase suggests a role of Fru-2,6-P2 in dormancy maintenance in cereal seeds. Alteration of gene expression due to imbibition independently of dormancy, involves changes in metabolism as shown by an in silico analysis of the barley transcriptome based on EST (Zhang et al., 2004), and detoxification processes. Epigenetic regulation may also be important during germination as illustrated by HV44A, a SET domain protein (Alvarez-Venegas and Avramova, 2000). Most significantly, the differential gene expression observed in dormant and non-dormant imbibed seeds reveals, in most cases, signalling elements. HV12D, showing similarity with an ARIADNE gene, might be implicated in ABA signalling as has been reported for the Arabidopsis germination process (Smalle et al., 2003). The expression of these TDF in embryos of barley seeds, the dormancy of which is modulated by various environmental conditions, is currently being characterized.


   Acknowledgements
 
The authors wish to thank Philippe Grappin (INA-PG, UMR Biologie des Semences, Paris, France), Marie-Christine Morere-Le Paven (UMR Physiologie Moléculaire des Semences, Angers, France), and Marie Barret (University College, Cork, Ireland).


   Abbreviations
 
ABA, abscisic acid; AdoMet, S-adenosyl methionine; cDNA-AFLP, complementary DNA-Amplified Fragment Length Polymorphism; D, dormant; EST, Expressed Sequence Tag; Fru-2,6-P2, fructose-2,6-bisphosphate; Met, methionine; ND, non-dormant; TDF, Transcript-Derived Fragment.


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