Journal of Experimental Botany, Vol. 55, No. 394, pp. 77-87, January 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
LEC1, FUS3, ABI3 and Em expression reveals no correlation with dormancy in Arabidopsis
Received 23 June 2003; Accepted 3 October 2003
1 Division of Cell and Molecular Biology, Biological Institute, University of Oslo, PO Box 1031, Blindern, N-0315 Oslo, Norway
2 Department of Botany, University of Georgia, Athens, GA 30602, USA
* Present address and to whom correspondence should be sent: Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. Fax: +47 22 93 44 40. E-mail: lars.baumbusch{at}klinmed.uio.no
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Dormant Arabidopsis seeds require stratification and light for germination. To study gene expression during establishment, maintenance and release of dormancy, various Arabidopsis ecotypes that are different in their degree of dormancy were investigated; three nsm mutants that lack the stratification-dependency, and the precocious germination and reduced dormancy of the abi3-1 mutant (insensitive to ABA). Genes examined by mRNA abundance include LEC1, FUS3 and ABI3, transcription factors that are major regulators of embryo development and, at least indirectly, play some role in the control of dormancy. Moreover, the late embryogenesis marker genes, AtEm1 and AtEm6, were examined in relation to the state of dormancy. The expression of LEC1, FUS3 and ABI3 mRNA is only marginally different during seed development in various strong or moderate dormancy wild types, nsm mutants and abi3-1. Therefore, it is unlikely that these transcription factors directly control the establishment of dormancy in Arabidopsis. Sole and various combina tions of light, temperature, and after-ripening regimes that alter germination behaviour were examined to determine if the expression of ABI3, AtEm1 and AtEm6 mRNAs were correlated with dormancy-breaking processes. ABI3 expression is influenced by cold and light, in a similar way in both dormant and non-dormant wild-type seeds. ABI3 transcript abundance in the nsm1 and nsm2 mutants is higher and in the nsm5-1 mutant is marginally lower than in wild-type seeds, but changes due to temperature and light factors are very similar to those that occur in wild-type seeds. The abundances of AtEm1 and AtEm6 mRNAs are equally affected by imbibition and cold temperature in mature and after-ripened seeds. The LEA transcript abundances for AtEm1 and AtEm6 are reduced in nsm mutants in a common, ABI3-independent pathway.
Key words: ABI3, Arabidopsis thaliana, Em genes, FUS3, gene expression, germination, late embryogenesis, LEC1, mRNA, seed development, seed dormancy.
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Introduction |
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The embryos of many seed plants have the remarkable property that they are able to go into a period of dormancy, which is characterized by the inability to germinate immediately in otherwise supportive conditions until specific environmental stimuli break or relieve this inhibition of germination. Despite many genetic and molecular studies of factors involved in embryogenesis and seed development (see reviews by Bewley, 1997; Galau et al., 1991; Goldberg et al., 1989; Hilhorst and Toorop, 1997; Kermode, 1990; Li and Foley, 1997; McCarty, 1995), the major genes controlling mature seed dormancy exclusively have not been identified.
Germination of fresh Arabidopsis seeds requires light and a cold treatment (stratification), which is an imbibition at 4 °C for several days. Dormancy is not broken by cold treatment of dry Arabidopsis seed alone; the cold-signal response requires that the seed be hydrated. Extensive dry storage results in seed ageing (after-ripening), after which germination occurs in less restrictive imbibition conditions (Hilhorst and Karssen, 1992). Various Arabidopsis ecotypes can be differentiated in their stratification and initial primary dormancy grade. INSOMNIAC (NSM) mutant alleles, lacking the stratification-breakable component of mature seed dormancy, have been isolated in Arabidopsis (Baumbusch, 2001). The mature seeds of nsm mutants are inhibited by exogenous abscisic acid, they retain the normal light-breakable component of dormancy and the plants have normal vegetative and reproductive growth (Baumbusch, 2001). Arabidopsis mutants with reduced dormancy (rdo), but otherwise wild-type behaviour, have been described by Léon-Kloosterziel et al. (1996), but these mutants show a large variation in the mutant and wild-type phenotypes. The sequences and characteristics of the RDO genes are still unknown.
The viviparous-1 (vp-1) mutant in maize (McCarty et al., 1989; Neill et al., 1987) and abscisic acid-insensitive (abi) mutants in Arabidopsis (Finkelstein, 1994; Koornneef et al., 1984) are capable of germinating in the presence of normally inhibitory concentrations of ABA. The Arabidopsis abi mutants have severe alterations including reduced dormancy (Finkelstein, 1994; Koornneef et al., 1984; Nambara et al., 1994, 1992; Ooms et al., 1993; Parcy et al., 1994). The studies of vp1 and abi3 mutants, focusing on late embryo functions such as desiccation tolerance, have not yet determined why the mutants are so non-dormant, but it is suggested that ABA is involved (Nambara et al., 1992; Ooms et al., 1993). ABA is probably involved in initiating late embryogenesis processes, but it is unlikely to be the exclusive major trigger for controlling the different aspects of seed development and dormancy (Bewley, 1997; Galau et al., 1991; Giraudat et al., 1994; Rock and Quatrano, 1995).
A number of mutants have been isolated with disturbed embryonic development programmes including perturbed dormancy establishment. For example, leafy cotyledon (lec) mutants of Arabidopsis are unable to distinguish between embryonic and vegetative patterns of plant development. Lec mutations exhibit morphological characteristics such as altered cotyledon morphology, desiccation intolerance and occasional vivipary (Meinke, 1992; West et al., 1994). The LEC1 gene encodes a transcription factor exclusively accumulating during seed development (Lotan et al., 1998). The fusca3 (fus3) mutation of Arabidopsis affects several aspects of embryogenesis, provoking a lec-like phenotype with ectopic trichomes, desiccation intolerance and precocious germination (Bäumlein et al., 1994; Keith et al., 1994; Nambara et al., 2000). The FUS3 and the ABI3 gene encode transcription factors that are similar to those encoded by the VP1 gene (Giraudat et al., 1992; Luerssen et al., 1998; McCarty et al., 1991). LEC1, FUS3 and ABI3 are postulated to play an important role in controlling mid- to late embryogenesis (Bäumlein et al., 1994; Castle and Meinke, 1994; Keith et al., 1994; Nambara et al., 2000; Vicient et al., 2000; West et al., 1994) and the establishment and maintenance of dormancy (Parcy et al., 1997). Studies of the wild type and abi3 fus3 double mutants suggest a negatively regulating function for ABI3 and FUS3 genes during late embryogenesis, possibly the inhibition of a particular set of genes during late embryo development (Nambara et al., 2000).
LEA (Late Embryogenesis-Abundant) genes are expressed late in embryogenesis (Galau et al., 1987, 1991; Hughes and Galau, 1991). LEA gene expression has been used as a convenient marker for altered expression in dormancy mutants or in mutations which affect dormancy. In Arabidopsis, the Em-like class I LEA genes, AtEm1 and AtEm6, have been cloned and characterized (Finkelstein, 1993; Gaubier et al., 1993). The fus3 and the lec1 mutants show altered expression of AtEm1 and AtEm6 mRNAs (Vicient et al., 2000).
An analysis of the establishment, maintenance and breaking of dormancy at the physiological and gene expression level using known gene expression markers is reported here. In order to elucidate genes supposed to be involved in the establishment of dormancy, the abundance of LEC1, FUS3 and ABI3 mRNAs in various Arabidopsis ecotypes during silique development was determined. Furthermore, gene expression changes of ABI3 and the LEA genes, AtEm1 and AtEm6, in nsm mutants and the Ws wild type during variable conditions have been studied to investigate the impact of temperature, light and after-ripening time on the maintenance and breaking of dormancy.
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Plant material
The Ws (Wassilewskija) ecotype of Arabidopsis thaliana was obtained from David Meinke (Department of Botany, Oklahoma State University, Stillwater, OK). The ecotype Cvi was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH). Ecotype C24 and mutant line abi3-1 (in Ler background) were obtained from T Palva (Helsinki, Finland). Seeds containing the ABA-insensitive3 (abi3-1) mutant allele, in Ler background, are generally considered to be non-dormant (Koornneef et al., 1989; Ooms et al., 1993).
The nsm mutants nsm1, nsm2 and nsm5-1 were isolated on the basis of their rapid germination in light, prior to stratification, and their phenotypes operationally defined as INSOMNIAC (Baumbusch, 2001). They are in Ws background and those used here were backcrossed at least once to Ws. The nsm5 mutants are hypersensitive to ABA, but appear normal in all other aspects of plant growth and development. Four nsm5 mutant alleles have been described and two additional mutants nsm1 and nsm2 have been characterized (Baumbusch, 2001). Complementation and physical mapping studies show that the nsm1, nsm2 and nsm5 mutants are in different genes at separate positions.
Growth and harvesting conditions
Plants were routinely grown in environmentally-controlled growth rooms (22 °C, 8/16 h dark/light, 100 µE m–2 s–1). For germination experiments, the plants were grown under continuous cool-white fluorescent light (50 µE m–2 s–1) at 20±2 °C. For developmental experiments, staging of developing siliques was performed by tagging individual flowers on the day of pollination, which was defined as the day the petals were visible. Only flowers borne on primary inflorescences were used and siliques were collected during 0–18 days after pollination (dap) of development. Under growth chamber conditions the seeds reached the mature stage at 15–16 dap. For each stage, a pool of siliques was harvested from 24 plants (0–5 dap), 12 plants (6–15 dap) and 24 plants (15–18 dap). Siliques were harvested and immediately frozen in liquid nitrogen (Parcy et al., 1994).
Germination conditions
After incubation treatments, the pooled seeds were collected in a tube and immediately frozen in liquid nitrogen and stored at –80 °C.
The seeds were sown on 1x MS inorganic medium (Murashige and Skoog, 1962) containing 2.5 mM 2-N-morpholinoethanesulphonic acid (MES), adjusted to pH 5.7 by the addition of KOH. Phytagel (Sigma) was added to 0.45% (w/v) as a solidifying agent prior to autoclaving. The autoclaved medium was poured into polystyrene plates. Seeds were sprinkled onto plates without prior sterilization. For incubation at room temperature and under light, the seeds were placed under standard germination conditions with growth room light standards (22 °C, 8/16 dark/light, 100 µE m–2 s–1). The plates of sown seeds were scored for germination daily by indicating on the bottom of the plate beneath each seed the day on which germination occurred. After 6 d, the plate was removed to 4 °C for 4 d to stratify non-germinated seeds, and the plates were scored for germination upon return to 22 °C. For incubation at a low temperature under light, the seeds were placed in a cold room at 4 °C under standard light conditions (8/16 h dark/light, 100 µE m–2 s–1). For incubations in the dark, the plates containing the seeds were wrapped under complete darkness in two layers of aluminium foil and placed in a light-proof box within a few seconds after sowing. The wrapped plates were transferred to the appropriate temperature conditions at 22 °C or 4 °C, respectively. Standard stratification, when done, was at 4 °C for 4 d.
For after-ripening experiments, mature seeds of the Arabidopsis wild-type ecotypes Ws, C24 and Cvi were placed on phytagel plates at weekly intervals and germination was measured over a period of 4 weeks.
Following germination testing, the seeds were stratified for up to three cycles to examine the stratification requirements for germination and the viability of the seed.
For stratification and after-ripening experiments, seeds were staged as follows. (a) Mature seed: fresh mature seeds, dark-brown and completely dry; siliques dry and yellow-brown. Seeds reached this stage after about 15–16 d post-pollination. The seeds were mature, but not after-ripened. (b) Mature seeds after-ripened for 4 weeks: mature seeds were removed from siliques and stored for 4 weeks in air at room temperature and under room light. Mature seeds were taken from eight plants and pooled together as a single sample and a minimum of 30–50 µg seeds for mature (0 weeks) or 4-week samples was aliquotted from the same pool of seeds. After each treatment, the seeds were frozen in liquid nitrogen and kept at –80 °C. The treatments for seeds of both ages included no incubation, incubation at 4 °C or 22 °C, and incubation in dark or light as described above.
RNA analyses
RNA was extracted using a modified protocol described by Downing et al. (1992). Approximately 0.1–1 g fresh weight of tissue, frozen in liquid N2, was transferred to a glass homogenization unit containing 1 ml lysis buffer (100 mM TRIS-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, pH 8.0, 1% LiDS, 1 mM DTT). The tissue was allowed to thaw during the 0.5–2.5 min homogenization. 900 µl phenol-chloroform-isoamyl alcohol (25:24:1 by vol.) was added to 900 µl of the extract. The solution was mixed for 30 s by vortex and incubated for 5–15 min on ice. After centrifugation at 11 500 rpm, for 15 min (this and all the following centrifugations were performed at 4 °C), the upper (aqueous) layer was collected and a second extraction with phenol-chloroform-isoamyl alcohol (25:24:1 by vol.) was performed followed by a final extraction with 1 vol. chloroform. For total RNA precipitation, one-third vol. 8 M LiCl was added to 2 M LiCl and incubated on ice overnight. After centrifugation at 9000 rpm, for 30 min, the RNA pellet was resupended in 100 µl TE (10 mM TRIS-HCl, 1 mM EDTA, pH 8). For the second RNA precipitation, 150 µl 5M KAc, pH 6, was added and left for 3–5 h on ice. RNA was collected by centrifugation at 9000 rpm for 30 min and redissolved in 100 µl TE. For the ethanol precipitation, 200 µl ETOH and 10 µl 3 M NaAc, pH 5.2, were added and the mixture incubated at –20 °C for 30–120 min. After centrifugation at 12 000 rpm for 30 min, the pellet was washed once with 70% EtOH and centrifuged for 5 min at 12 000 rpm. The drained pellet was dried under vacuum. RNA was allowed to dissolve for 5 min at 60 °C in 30 µl TE with periodic vortexing. RNA concentration was estimated by absorbance at 260 nm of a suitably diluted 2 µl aliquot.
Total RNA (10 µg) was size-fractionated on a 1.7% agarose, 3% formaldehyde gel (Sambrook et al., 1989), transferred to nylon filters (Hybond-N, Amersham) by capillary action and afterwards UV-cross linked with 120 000 mJ cm–2 (Hoefer system). Filters were hybridized at 68 °C with AtEm1, AtEm6, LEC1, FUS3, and ABI3 probes according to the method of Church and Gilbert (1984) and at 70 °C with a rDNA probe to verify that equal amounts of RNA were present in each line. Filters were stripped with 1 mM EDTA, pH 8.0, 0.1% SDS for subsequent rehybridization. Autoradiographic signals were scanned and quantified. 32P-labelled DNA probes from PCR reactions were generated in a random-priming reaction with biotinylated single-stranded template bound to magnetic streptavidin-coated beads (Dynabeads M280-streptavidin, Dynal Biotech AS, Norway and GenoPrepTM Streptavidin beads, Genovision, Norway) as described (Espelund et al., 1990). The AtEm1, AtEm6, LEC1, FUS3, and ABI3 probes were generated in a standard random-priming reaction using a Random Primed DNA labelling kit (Roche, Switzerland). AtEm1 (454 bp) primers: AtEm1-f 5'-GTCCGTA TCTGTGTCGGAAC-3' and AtEm1-r 5'- CACTGCCGTTAGTAC AAGCC-3'; AtEm6 (279 bp) primers AtEm6-f 5'-CGTATGAC GTACCGATTGTC-3' and AtEm6-r 5'-GGTCATCACCATCCLEC1 (626 bp) primers LEC1-f –5'-GTATCGTGGTCCAGCAGC AA-3' and LEC1-r 5'-GGATTCATCTTGACCCGACG-3'; FUS3 (927 bp) primers FUS3-f 5'-CTCATGGTCTGCAGCTAGGTG-3' and FUS3-r 5'-CGAATGTTCCGAACTTGGAG-3'; ABI3 (2162 bp) primers ABI3-f 5'-GGATGGAATTGATGAAGTTG-3' and ABI3-r 5'-GCATGTCTCCACCACTGTTA-3'. The rDNA probe was generated using a biotinylated single-stranded template bound to streptavidin-coated beads in a specific priming reaction (Stacy et al., 1991), rRNA (1803bp) NS3-F 5'-GCAAGTCGTGTGCC AGCAGCC-3' and NS4-r 5'-CTTCCGTCAATTCCTTTAAG-3'.
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Degree of dormancy in wild-type ecotypes
The Ws ecotype exhibits only moderate seed dormancy. Therefore, the germination of other Arabidopsis ecotypes was examined over an after-ripening period of 4 weeks to identify ecotypes having higher levels of dormancy. The results of this survey are reported in Table 1. The Arabidopsis ecotypes, Cvi, C24 and Ws, are completely dormant as fresh mature seeds. The germination of seeds of the Ws ecotype increased during the 4-week test period to a final germination of 97% after 4 weeks of after-ripening. By contrast, Cvi and C24 seeds remain dormant even after 4 weeks of after-ripening. As another indicator of dormancy strength, germination was assayed after stratification treatments. The number of stratification treatments required to completely break dormancy in mature seeds is ecotype-dependent. For fresh seeds of the Ws ecotype, a single immediate stratification is sufficient to break dormancy completely, resulting in a 100% germination within 4 d upon return to room temperature conditions (Table 1). Thus, the Ws wild type can be classified as moderately dormant. By contrast, fresh mature seeds of Cvi required two cycles of stratification punctuated by 2 d at room temperature before dormancy was broken. On the basis of both the time requirement for after-ripening and the immediate stratification demand of mature seeds, the three ecotypes tested can be rated as Ws<<C24<Cvi, from moderate to highly dormant.
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Expression of LEC1, FUS3 and ABI3 mRNAs during silique development
Investigations of developmental differences in the accumulation kinetics of LEC1, FUS3 and ABI3 mRNAs of the ecotypes Cvi, C24, Ws and the nsm1, nsm2, nsm5-1, and abi3-1 mutants were performed using developmental northern blots of total silique RNA (Fig. 1). All three mRNAs have been found to be expressed only in embryos (Bäumlein et al., 1994; Giraudat et al., 1992; Lotan et al., 1998; Nambara et al., 2000, 1995; West et al., 1994). Each probe detected a single band, as expected. Hybridization with gene specific probes was followed by hybridization with an 18S rDNA probe to detect any RNA loading differences. No major differences in RNA loading were noted (Figs 2, 3; for Fig. 1 data not shown). LEC1 transcript expression was detected at low intensity throughout silique development. The different ecotypes, Cvi, C24 and Ws, show the highest LEC1 mRNA expression at 4–7 dap (Fig. 1). Compared with the Ws wild type, the nsm1 accumulates transcript a day earlier, beginning at 3 dap. By contrast, nsm5-1 has high transcript abundance for at least a day longer than the other ecotypes and mutants, from 3–8 dap. The abi3-1 mutant shows weaker accumulation through at least 5 dap, with a maximal expression at 7 dap (Fig. 1). FUS3 transcripts accumulate later during silique development. In Cvi, C24 and Ws FUS3 transcript increases at 8 dap, lasting until 14 dap for Cvi and C24 and until 13 dap for Ws. All the mutants begin to accumulate FUS3 at a slightly earlier time. The nsm1 and nsm2 mutants begin to accumulate FUS3 2–3 d earlier, and nsm5-1 and abi3-1 about 1 d earlier than the wild types. High FUS3 transcript expression is terminated by 14 dap in all ecotypes and mutants (Fig. 1). Detectable ABI3 mRNA expression starts at about 5 dap, with a pattern of increasing abundance during silique development with a maximal accumulation at 19 dap. All the wild types and the nsm1, nsm2 and nsm5-1 and abi3-1 mutants show a similar abundance of the ABI3 transcripts, however, nsm5-1 may accumulate transcript at higher concentrations through 19 dap than the others (Fig. 1). Given the potential uncertainties in the age of the siliques used for RNA preparations, it was concluded that differences in the rate of development are insignificant among genotypes, and that no obvious correlation between developmental expression and dormancy degree of the mature seeds emerges.
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AtEm1, AtEm6 and ABI3 gene expression in mature and after-ripened seeds
Total RNA from the ecotypes Cvi, C24, Ws, the nsm-mutants, nsm1, nsm2, nsm5-1, and the abi3-1 mutant was extracted from the mature seeds and from the mature seeds that had been after-ripened for 4 weeks. For the wild types and mutants, only marginal transcript concentration differences are detectable between mature and after-ripened seeds. All nsm mutants and the abi3-1 mutant show about half of the AtEm1 and AtEm6 transcript accumulation compared with the Ws wild type (Fig. 2). The C24 and Ws ecotypes display similar AtEm1 and AtEm6 transcript amounts, with a 2-fold higher abundance than the Cvi ecotype (Fig. 2). C24 seeds show greater ABI3 content than the other ecotypes, 2-fold higher than Ws and 4-fold higher than Cvi (Fig. 2). All the nsm mutants have, as mature or after-ripened seeds, a 1.5–2-fold higher ABI3 expression quantity than the Ws wild type. Both the Ws wild type and all nsm mutants have half the ABI3 transcript amount in after-ripened seeds compared with the mature seeds. The abi3-1 mutant shows a high ABI3 accumulation at mature and after-ripened stage (Fig. 2). The results of the gene expression studies reported in Fig. 2 indicate that the differences in AtEm1, AtEm6 and ABI3 abundance of the various ecotypes and mutants are not related to differences in dormancy levels.
Influence of light, temperature and after-ripening time on AtEm1, AtEm6 and ABI3
Dormant wild-type Arabidopsis seeds require a combination of imbibition, temporally defined cold stimulus (stratification) and light to break dormancy. LEA genes might be involved in the inhibition pathway of germination during the later stages of development. Transcript accumulations of AtEm1, AtEm6 and ABI3 were investigated under variable physiological conditions in order to investigate a possible functional relationship between LEA genes and seed dormancy. Mature Ws seeds remain dormant after a 4 d incubation at room temperature in light or darkness, but after-ripened Ws seeds germinate immediately (Table 1; data not shown). Mature seed dormancy in the Ws ecotype is broken by a cold incubation at 4 °C for 4 d (Table 1). All nsm mutants germinate to 100% without prior stratification (Baumbusch, 2001). Despite the lack of stratification dependency, the light requirement for germination in nsm mutants is still present. Thus, nsm mutants are only affected in the stratification-breakable, but not in the light-perceptible, part of seed dormancy (Baumbusch, 2001).
After-ripening, humidity, temperature, and light influence the state of dormancy in mature Ws wild-type seeds (Table 2). The influence of these factors on transcript abundances were determined in Ws wild-type seeds and compared with non-dormant nsm1, nsm2 and nsm5-1 mutants using mRNAs probes corresponding to AtEM1, AtEm6 and ABI3 (Fig. 3). Equal loading was tested with ribosomal RNA detection using radioactive 18S rDNA probe.
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Mature and dormant Ws wild-type seeds (M1) have about one-third lower AtEm1 and AtEm6 transcript concentrations than the mature seeds imbibed at 22 °C in darkness (M2; see Fig. 3). The stratification treatment of mature seeds incubated for 4 d at low temperature (4 °C) in darkness (M3) or light (M4) results in a dramatic reduction in AtEm1 and AtEm6 expression compared with mature seeds (M1) or with mature seeds which have been incubated at 22 °C in the dark (M2). Remarkably, an equal reduction in LEA gene transcripts is visible in after-ripened seeds. After-ripened non-dormant (A1) seeds have about one-third lower AtEm1 and AtEm6 transcript amounts than in after-ripened seeds imbibed at 22 °C in darkness (A2). Moreover, after-ripened seeds, incubated at 4 °C in dark (A3) or light (A4), have less than a quarter AtEm1 and AtEm6 mRNA accumulation than that observed in after-ripened seeds (A1) or in after-ripened seeds which have been imbibed at 22 °C in darkness (A2). For both cases of mature and after-ripened wild-type seeds, LEA gene transcript abundances are substantially lower after 4 °C incubation in light (M4 or A4) compared with seeds incubated at 4 °C in darkness (M3 or A3). These results indicate that light has a modulating effect on LEA gene transcription abundance in Ws wild-type seeds (Fig. 3).
In mature (M1), after-ripened (A1) or in mature and after-ripened seeds which have been incubated at 22 °C in darkness (M2 and A2), AtEm1 accumulations in Ws wild-type seeds are equal to or higher than in all nsm mutants. Under similar conditions AtEm6 accumulations in the wild type are 2–7-fold higher than in all nsm mutants. These higher abundances occur in both mature (M1) and mature incubated at 22 °C in darkness seeds (M2), as well as in after-ripened (A1) and after-ripened incubated at 22 °C in darkness seeds (A2). Remarkably, a strong increase in transcript abundance in mature nsm2 seeds is seen at 4 °C cold incubation in darkness (M3), 7-fold for AtEm1 and 14-fold for AtEm6, but not during cold incubation in light (M4). In summary, wild-type seeds have a lower LEA gene expression during cold temperature incubation, but equally for mature (dormant) and for after-ripened (non-dormant) seeds. The nsm mutants, compared with the wild type, generally have decreased LEA mRNA accumulation.
ABI3 mRNA accumulation in mature wild-type seeds incubated at 4 °C in darkness (M3) is 10-fold higher compared with the expression in mature seeds (M1) as well as in seeds which have been incubated at 22 °C in darkness (M2). Similarly, after-ripened (A3) wild-type seeds incubated at 4 °C in darkness show an approximately 10-fold enhanced ABI3 mRNA accumulation than the after-ripened seeds (A1) or after-ripened seeds incubated at 22 °C in darkness (A2). As was observed for the LEA gene expression, the presence of light modifies the temperature dependence of transcript concentrations. Mature wild-type seeds incubated at 4 °C in darkness (M3) have twice the ABI3 abundance of 4 °C plus light-incubated seeds (M4) and four times the ABI3 abundance of seeds which have been incubated in 22 °C plus light (M5). Compared with the wild-type, ABI3 transcript concentrations in nsm1 and nsm2 mutants are higher in mature or after-ripened seeds at 4 °C incubation in darkness or light (M3, A3 and M4).
The results show that ABI3 gene expression is induced equally by cold temperature incubation in darkness and in mature and after-ripened seeds of wild-type genotypes. This induction is even stronger in nsm1 and nsm2. Thus, there are no clear dormancy-related differences in expression of ABI3.
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Characterizations of Arabidopsis plants from different geographical regions and ecosystems suggest that there is considerable genetic variation (reviewed in Alonso-Blanco and Koornneef, 2000). Early observations of seed germination established that there were differences in stratification dependence, after-ripening time, and storage conditions among different Arabidopsis ecotypes (Kugler, 1951; Laibach, 1951; Langridge, 1957). The Arabidopsis ecotypes, Cvi, C24 and Ws, which were not included in these early studies, were tested. C24 and Cvi are completely dormant as mature and after-ripened seeds and Ws seeds are dormant as mature seeds, but germinate 100% after 4 weeks of after-ripening. Based on their germination frequency and stratification requirements, the tested ecotypes can be ranked from moderately to strongly dormant, i.e. Ws<<C24<Cvi. This ranking complements a recent investigation of Ws versus the Ler, Col and En ecotypes (Debeaujon et al., 2000). Cvi is a strongly dormant ecotype and therefore has been used for creating recombinant inbred lines derived from crosses with the low-dormancy Ler ecotype for analysis of quantitative trait loci affecting dormancy (Alonso-Blanco et al., 2003; van der Schaar et al., 1997).
The interaction of three physiological factors, age of seed, incubation of imbibed seed at low temperature (of an ecotype-specific duration), and the correct quality and quantity of light, is necessary for breaking dormancy in wild-type Arabidopsis. No single one of these factors breaks dormancy, but after-ripening reduces the requirement for stratification (Table 1). The expression of the well-characterized regulatory genes, LEC1, FUS3, and ABI3, and the post-abscission-abundant LEA genes, AtEm1 and AtEm6, were investigated. Transcript abundance was investigated in the moderately dormant Ws ecotype versus the strongly dormant Cvi and Col ecotypes, as well as in nsm mutants lacking the stratification requirement for germination.
Under no conditions could dormancy-dependent gene expression differences between strongly dormant and moderately dormant ecotypes be detected in mature or after-ripened seeds. Seeds of the aba-1 and abi3-4 mutants are reported to have reduced AtEm1 and AtEm6 accumulation (Parcy et al., 1994) and mutant seeds of abi3-1 have reduced AtEm6 accumulation (Finkelstein, 1993). It has been shown here that the AtEm1 accumulation of abi3-1 mutant seeds is also reduced. The developmental mutants lec1 and fus3 show similar altered LEA gene expression (Vicient et al., 2000). The expression in lec1-1 and lec1-2 in mature seeds at different stages of development has been described as being reduced for AtEm6 for both mutants compared with the wild-type and being unchanged for AtEm1 (Vicient et al., 2000). The results presented here show that all nsm mutants have lowered AtEm1 and AtEm6 expression in mature and after-ripened seeds compared with the wild type with the exception of an extremely high induction by stratification of AtEm1 and AtEm6 in nsm2 with respect to the other mutants and wild types. The reduced LEA abundance in nsm mutants could be explained by lower than normal transcription. Consequently, since three different nsm mutants show a similar reduction in LEA transcripts concentration, the lesions must affect different components in the regulatory pathway of at least the LEA genes. This might reflect that nsm mutants are skipping a developmental stage that involves preparation for dormancy. However, this preparation cannot involve desiccation protection, because nsm dry seeds are viable. In these experiments, the amount of LEA transcripts are increased by imbibition and reduced by cold temperature, with a light-induced modulation effect. Their induction during imbibition could to be a hydration effect or just preparation for dormancy, since it is an open question if dormancy is imposed upon imbibition rather than after abscission or during drying. The observation of reduced LEA mRNA accumulation after a 4-week after-ripening time in Cvi and Col ecotypes, suggests that LEA gene expression in mature seeds is a useful marker for dormancy and it is possible that LEA gene expression is associated with dormancy regulation pathways. However, involvement of these LEA genes with desiccation protection is unlikely. First, dry seeds of the mutants are viable. Second, LEA expression is considerably lessened in the mature seeds of the mutants and declines in the nsm mutants upon imbibition.
The three genes LEC1, FUS3 and ABI3 have been found to be general regulators for different aspects of seed development between mid to late embryogenesis (Bäumlein et al., 1994; Parcy et al., 1997; West et al., 1994) including dormancy (Parcy et al., 1997). LEC1 mRNA accumulation starts at preglobular to bent cotyledon stage, is limited to seed development, and is high during seed maturation (Lotan et al., 1998; West et al., 1994). In this study, LEC1 transcripts were much higher in abundance at 3–7 dap than subsequently at 8–16 dap. With these experimental conditions, FUS3 expression was observed during mid-embryogenesis and ABI3 expression was observed in mid- to late embryogenesis for the Ws, C24 and Cvi ecotypes (Fig. 1). Earlier investigations of FUS3 expression for the Dijon-G or Col ecotypes (Luerssen et al., 1998) and ABI3 expression for the Ler ecotype (Parcy et al., 1994) coincide with this temporal accumulation.
Different Arabidopsis ecotypes show variation in their dormancy behaviour and in their expression of the two LEA mRNAs in mature seeds, but not in the temporal mRNAs accumulation during silique development of the transcription factors LEC1, FUS3 and ABI3 (Fig. 1). This observation suggests that these transcription factors are not the major immediate regulators required for the establishment of dormancy. In nsm mutants, which lack stratification-breakable seed dormancy, the abundance of LEC1, FUS3 and ABI3 mRNAs are not, or only marginally, changed during development compared with the Ws wild type. It therefore seems unlikely that these transcription factors are the cardinal triggers for the regulation of stratification-breakable dormancy in Arabidopsis.
The nsm mutants show decreased AtEm1 and AtEm6 mRNA abundance, but generally increased ABI3 mRNA abundance compared with the wild type. ABI3 mRNA abundance in nsm seeds is especially high after imbibition in the cold, under conditions that break dormancy in wild-type seeds. However, similar cold-related alterations in ABI3 mRNA are observed in wild-type mature dormant and after-ripened non-dormant seeds when imbibed. ABI3 mRNA accumulations undergo similar after-ripening age-related changes in the nsm mutants and the wild type. In nsm mutants, the strongly cold-induced ABI3 gene expression is reduced by light. These results suggest that the ABI3 gene is regulated by a cold- and a light-modulated mechanism not directly related to the state of dormancy. In addition, the non-dormant nsm mutants show the strongest ABI3 expression during cold incubation. Further investigations will show if cold-induced genes such as the SFR genes (Thorlby et al., 1999) are influenced by NSM genes or vice versa. The nsm mutants have normal vegetative and reproductive growth, yet only marginal changes in the expression of LEA genes and embryo-regulating genes so far examined. However, the variety of ABI3 and LEA gene expression across the nsm mutants suggests that the particular genes have several distinct functions in a common dormancy-related pathway.
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We wish to thank SH Engebretsen and R Falleth for technical assistance. This work was supported by grant No. 114361/112 to KSJ from the Norwegian Research Council and to GAG from The University of Georgia Program in Biological Resources and Biotechnology.
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