Journal of Experimental Botany, Vol. 51, No. 347, pp. 995-1003,
June 2000
© 2000 Oxford University Press
Changes in gene expression in the leafy cotyledon1 (lec1) and fusca3 (fus3) mutants of Arabidopsis thaliana L.
Laboratoire de Physiologie et Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique UMR 5545, Université de Perpignan, Avenue de Villeneuve, 66860 Perpignan Cedex, France
Received 27 May 1999; Accepted 21 January 2000
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Arabidopsis thaliana L. leafy cotyledon1 (lec1) and fusca3 (fus3) mutants show multiple phenotypic defects during seed development. In this report the effects of these mutations are examined at the molecular level. The patterns of protein accumulation in lec1 and fus3 seeds are severly altered. In lec1 seeds the steady-state mRNA levels of several late embryogenesis genes were reduced. Different patterns of expression were observed, indicating the occurrence of several regulatory pathways. The effect of lec1 mutations on the expression of the late-embryogenesis abundant AtEm1 gene was examined in detail. In lec1-1 seeds, the AtEm1 gene was expressed at a higher level than in the wild type and earlier in development. The activity of an AtEm1 promoter/ß-glucuronidase reporter gene construct in transgenic A.thaliana plants was studied. Changes in promoter activity in lec1-1 with respect to wild-type seeds were correlated with changes in corresponding mRNA steady-state levels. fus3-2 mutation produced similar changes in AtEm1 promoter activity as lec1-1, which is consistent with the hypothesis that LEC1 and FUS3 might act in the same regulatory pathway. Transgenic analysis using 5'–promoter deletions demonstrated that at least two regions of AtEm1 gene promoter interact with the LEC1-dependent transcriptional regulatory pathway. In spite of expression of the AtEm1 promoter and accumulation of AtEm1 mRNA, the corresponding Em1 protein does not accumulate in lec1-1 seeds. The ABA inducibility of the AtEm1 promoter was not affected by the lec1 mutation.
Key words: abi3, Em genes, gene expression, late embryogenesis, seed development.
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Introduction |
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Mutational approaches have begun to yield insights regarding the molecular mechanisms controlling seed development. ABA-insensitive mutations have demonstrated the role of this hormone in seed maturation (Leung and Giraudat, 1998
). However, other factors are clearly involved (de Vries et al., 1998; Harada et al., 1998; Wobus and Weber, 1999). One of the most severe alterations of seed maturation in higher plants has been observed in the leafy cotyledon (lec) mutants (Meinke, 1992). Three different mutants with a phenotype similar to the original lec1 mutant have been reported, lec1, lec2 and fus3 (Baumlein et al., 1994; Keith et al., 1994; Lotan et al., 1998; Luerßen et al., 1998; Meinke, 1992; Meinke et al., 1994; West et al., 1994). Leafy cotyledon1 (lec1) mutant embryos present strong morphological alterations after the torpedo stage. The lec1 embryos show reduced and almost transparent hypocotyls with highly vacuolated cells, rounded cotyledons that remain green unusually late in development and an abnormal accumulation of anthocyanin is often observed prior to desiccation (Meinke, 1992; Meinke et al., 1994; Parcy et al., 1997; West et al., 1994). The cotyledons of lec1 embryos have a vascular system intermediate between normal cotyledons and leaves, having mature xylem elements and stomata that, in wild-type embryos, appear only after germination. The lec1 seeds are desiccation intolerant and occasionally viviparous at maturity. The lec1 embryos can germinate and give rise to morphologically normal plants only if they are harvested before seed desiccation. The plants produced are viable and phenotypically normal except for the presence of leafy cotyledons with trichomes on the adaxial surface and the production of 100% defective seeds. The mutant embryos contain less protein and lipid bodies than in the wild-type embryos (Meinke et al., 1994). lec1 embryos present defects in suspensor morphology during early embryogenesis (Lotan et al., 1998). Two alleles of the lec1 mutant have been identified, lec1-1 and lec1-2. Both alleles show very similar phenotypes, general morphology, internal anatomy, and response in culture. The lec1-1 mutation is the result of a deletion overlapping the lec1 gene and lec1-2 was produced by the insertion of a T-DNA 115 bp upstream from the lec1 coding region (Lotan et al., 1998). It is likely that lec1-1 could correspond to a null allele whereas it is conceivable that lec1-2 might be leaky. The lec1 gene has been cloned and the deduced protein shows sequence similarity with the HAP3 subunit of the CCAAT box-binding factor (CBF) (Lotan et al., 1998). lec1 is a member of a small gene family. lec1 mRNA accumulation is restricted to seed development from preglobular to bent cotyledon stage and decays during seed maturation.
Another mutant, fusca3 ( fus3), has a similar phenotype to lec1-1 and lec1-2, but the cotyledons are not so heavily altered in shape (Baumlein et al., 1994; Keith et al., 1994; Meinke et al., 1994; Miséra et al., 1994). fus3 seeds are paler than lec1 and the pattern of anthocyanin accumulation in the embryo is different. The fus3 embryos accumulate anthocyanin mainly in the junction between cotyledons and the hypocotyl whereas lec1 embryos accumulate anthocyanin mainly in the cotyledon margins. Moreover, trichomes in fus3 cotyledons are not as prominent as in lec1. Three fusca3 alleles have been isolated. fus3-1 and fus3-2 were identified by the accumulation of a purple pigment in late embryogenesis (Baumlein et al., 1994; Miséra et al., 1994), and a third allele, fus3-3, was identified by screening of immature mutagenized seeds for their ability to germinate during the green cotyledon stage (Keith et al., 1994). No significant differences between the alleles have been reported. The fus3 gene is a member of the ABI3/VP1 B3 domain protein family (Luerßen et al., 1998). A third mutation is leafy cotyledon2 (lec2) (Meinke, 1992). Alterations in the development of the lec2 embryos are not as severe as in lec1 and fus3. A single allele of lec2 has been reported (Meinke et al., 1994) and at present, the sequence and function of this gene are not known.
The complexity of lec1 and fus3 phenotypes indicates that LEC1 and FUS3 proteins may be required for the activation of a wide range of embryo specific genetic programmes. The steady-state mRNA levels of some storage protein and oleosin genes and their promoter activities are reduced in lec1 and fus3 embryos (Baumlein et al., 1994; Kirik et al., 1996; Parcy et al., 1997), but the promoter activity of some late embryogenesis genes is not severely affected in fus3 (Baumlein et al., 1994) and lec1-2 seeds (West et al., 1994). In lec1-2 embryos some genes prevalently expressed during post-germinative growth are active simultaneously with late-embryogenesis genes (West et al., 1994) and the accumulation of the MADS domain protein AGL15 is reduced in lec1-2 but not in lec1-1 embryos (Perry et al., 1996). The expression of MYB genes have been found to be altered in fus3 embryos (Kirik et al., 1998a, b).
Further molecular analysis in the leafy cotyledon mutants should help to elucidate the regulatory networks that function during embryogenesis and seed maturation in A.thaliana. As shown for the abi3 mutant (Parcy et al., 1994), monitoring a set of molecular markers should help to clarify the role of these regulatory loci in the control of seed development. This consideration prompted the authors to analyse the effect of the lec1-1 and lec1-2 mutations on the expression of several genes previously used as genetic markers of seed development.
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Materials and methods |
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Plant material and growth conditions
The lec1-1 seeds were obtained from Dr David Meinke (Oklahoma State University), lec1-2 from Dr John Harada (University of California, Davis) and fus3-2 from Dr Simon Miséra (IPK, Gatersleben). Transgenic seeds containing an AtEm1 promoter/ß-glucuronidase reporter gene chimeric construct (Hull et al., 1996
) were kindly provided by Gillian Hull (University of Perpignan), and plants containing ABI3 promoter/ß-glucuronidase reporter gene construct by Dr Jérôme Giraudat (ISV, Gif sur Yvette). Plants were grown in a greenhouse (22 °C, 16 h day). Homozygous lec1-1 mutant plants were obtained by plating surface-sterilized immature embryos on MS medium (Murashige and Skoog, 1962) and transferring to soil after 15 d of in vitro culture. All transgenic plants were grown according to the French safety guidelines and the French Genetic Engineering Commission approved these experiments.
Total RNA isolation and Northern hybridization
RNA extractions were performed from frozen material as previously described (Vicient and Delseny, 1999). 2 µg of total RNA were fractionated on 1.5% agarose/2.2 M formaldehyde gels as described previously (Farrell, 1993) and stained with ethidium bromide to ensure equal loading. RNA was transferred to Hybond N+ membranes (Amersham) by capillarity (Sambrook et al., 1989) and UV-fixed using a Stratagene Stratalinker. Membrane prehybridization was performed using 0.25 M NaHPO4 (pH 7.2), 7% SDS (p/v) and 1 mM EDTA to which 100 mg l-1 of previously denatured calf thymus DNA were added. After 20 min prehybridization at 65 °C, the DNA probe was added to the prehybridization buffer. Radioactive probes were generated using the Megaprime labelling kit (Amersham). Following overnight incubation, membranes were washed twice for 20 min at 65 °C with 20 mM NaHPO4 (pH 7.2), 1% SDS (p/v) and 1 mM EDTA. A last wash was performed for 10 min in the same buffer containing only 0.1% SDS and at the same temperature. Signal detection was carried out using Kodak X-ray film and Hyperscreen intensifying screens (Amersham). The filters were finally hybridized with a radish 18S rRNA probe (Grellet et al., 1989) as a control for RNA loading.
Protein extraction and analysis
Total protein was extracted from A. thaliana dry seeds using the following extraction buffer: 10 mM NaCl, 10 mM TRIS-HCl pH 8.3 1 mM TLCK (tosyl-L-lysine-chloromethylcetone from Sigma) as protease inhibitor. After centrifugation (20 min, 13 000 rpm), protein content was determined using the Bradford method (Bradford, 1976). Electrophoretic analysis, protein blotting and immunological detection were conducted as described previously (Bies et al., 1998). Proteins were visualized by staining with Coomassie Brilliant Blue R250.
Generation of crosses
For crosses, heterozygous mutant plants were used as female parents and homozygous transgenic plants carrying the promoter/ß-glucuronidase fusion as male parents. Closed flowers were stripped of sepals, petals and anthers just prior to stigma maturity. Two days later the stigma was brushed with anthers of the male parental plants. Crosses were checked by the absence of mutant seeds in the F1 population. 75% of the F2 population inherit the promoter/ß-glucuronidase fusion and 25% exhibit the mutant phenotype. Wild-type and mutant seeds of the F2 population of at least three transgenic crosses were assayed for GUS expression. Dry seeds of crosses were collected from the same pods to minimize developmental and physiological differences, mutant seeds were separated from wild-type seeds under a binocular microscope and frozen in Eppendorf tubes. For the analysis of GUS activity during seed development siliques were harvested from the same inflorescence. Seeds of consecutive siliques were pooled and mutant seeds were separated from wild-type seeds under a binocular microscope, transferred to Eppendorf tubes and frozen.
Measurements of GUS activity
Fluorometric GUS assays were performed as previously described (Devic et al., 1995). GUS activity was standardized with respect to protein content in the extract determined by the Bradford method (Bradford, 1976).
ABA induction
Homozygous lec1-1 and wild-type immature transgenic seeds were harvested at 12 d after pollination and incubated on solid MS medium (Sigma) supplemented with 50 µM of ABA (mixed isomers, Sigma). Control seeds were incubated in parallel on MS medium without ABA. Wild-type and mutant immature seeds were extracted from the same siliques to avoid developmental or environmental differences. Seeds were incubated during 24 h. GUS activity determinations were done in duplicate using 20 seeds per treatment obtained from 4 independent crosses.
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Effects of the lec1 mutations on gene expression in mature seeds
The levels of accumulation of different mRNAs in dry seeds of lec1-1 and lec1-2 mutants were determined by RNA blot analysis (Fig. 1
). The lec1-1 seeds were kindly provided by Dr David Meinke (Oklahoma State University) and lec1-2 by Dr John Harada (University of California, Davis). The parental ecotype of both mutant alleles, Wassilewskija, was included as a control. Nine cDNA probes were used corresponding to genes expressed during late embryogenesis. Each probe detected a single band on RNA gel blot analysis. AtEm1 and AtEm6 probes correspond to well-characterized A. thaliana class I lea (Late Embryogenesis Abundant) genes (Finkelstein, 1993; Gaubier et al., 1993), M10 and M17 correspond to a new type of lea genes (Raynal et al., 1999) and the other probes correspond to clones obtained from the systematic analysis of A. thaliana dry seed cDNA libraries (Cooke et al., 1996) with the following protein sequence similarities: PAP260 with the tomato lea25 gene (Cohen and Bray, 1992); PAP51 with the cotton lea gene d113 (Baker et al., 1988); PAP240 with the rapeseed Lea76 gene (Harada et al., 1989); PAP85 with a broad bean vicilin (Bassuner et al., 1987); and PAP137 with a protease inhibitor protein (Ogata et al., 1991). The accession numbers are given in the legend to figure 1. PAP260 and PAP51 belong to the same multigene family but do not cross hybridize. Filters were also hybridized as a control with a radish 18S rDNA probe (Grellet et al., 1989). The accumulation of PAP260, AtEm6, M10, M17, and PAP137 mRNAs was severely reduced in both mutants alleles compared to wild-type. PAP240 and PAP85 mRNAs abundance was also reduced although less severely. A different situation was found with PAP51 and AtEm1 probes. PAP51 mRNA accumulation was reduced in the two mutant alleles, but the reduction was stronger in lec1-2 than in lec1-1. AtEm1 mRNA level was increased in lec1-1 compared with the control, but in lec1-2 seeds the level was similar to the control. Similar results were obtained in three repetitions of this experiment using RNAs obtained in independent extractions.
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Effects of the lec1 and fus3 mutations on protein accumulation in mature seeds
The patterns of protein accumulation in wild-type, lec1-1, lec1-2, and fus3-2 seeds were compared. Total proteins were obtained from 50 lec1-1, lec1-2 and fus3-2 seeds. As controls, total proteins from 50 seeds of the corresponding wild-type ecotypes were also extracted (Wassilewskija for lec1-1 and lec1-2, and Dijon for fus3-2). For each sample, total proteins were extracted in the same volume of extraction buffer and equal volumes of the soluble fraction of each extraction were electrophoresed in a SDS-polyacrylamide gel and stained. The patterns of protein accumulation obtained in the mutant seeds were different from those observed in the wild-type, but roughly similar between the three mutants (Fig. 2). The intensity of many of the bands present in the wild-type seed extracts was substantially lower in the mutant seed extracts particularly those corresponding to seed storage protein. On the other hand, some bands not detectable or detectable at low intensity in the wild-type extracts appeared clearly in the mutant ones. The changes in the patterns of protein accumulation were similar to those previously observed for fus3 seeds (Kirik et al., 1996; Keith et al., 1994).
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Effect of the lec1-1 mutation on AtEm1 and AtEm6 gene expression in developing seeds
Expression of AtEm1 and AtEm6, two class I lea genes, was further investigated by examining accumulation kinetics of corresponding mRNAs during seed development in wild-type and lec1-1 siliques (Fig. 3). Wild-type and homozygous lec1-1 mutant plants were grown in parallel. Total RNA was extracted from siliques at different stages of development and used in RNA blot analyses. The accumulation of AtEm1 mRNA in wild-type plants was detected, in these experimental conditions, as early as 12 d after pollination (dap), with the greatest accumulation at about 17 dap as previously reported (Parcy et al., 1994; Bies et al., 1998). In contrast, in the lec1-1 mutant seeds, AtEm1 mRNA was already detected at 3 dap, much earlier than in wild-type seeds. At 7 dap the level of mRNA accumulation was similar to those observed in mature seeds. To control equal loading, the ribosomal RNA in the gel was stained with ethidium bromide and visualized under UV light. The same RNA blots were used to analyse the expression of the AtEm6 gene. In wild-type plants, AtEm6 mRNA starts to accumulate at 17 dap, with a maximum level in mature seeds, as expected. In the lec1-1 mutant the time-course of AtEm6 mRNA accumulation is similar to wild-type, but the steady-state level is strongly reduced.
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AtEm1 and AtEm6 protein accumulation in lec1-1 seeds
During seed development, AtEm1 and AtEm6 proteins accumulate at the highest level in mature seeds, as has been monitored by Western analysis using a specific immune serum (Bies et al., 1998). The immune serum used detected two protein bands with apparent molecular masses of 21 and 16 kDa in wild-type mature seed extracts. These values are higher than the calculated molecular weights and are due to delayed migration of Em proteins in SDS gels (Raynal et al., 1989). Total protein extracts were prepared from wild-type and lec1-1 seeds. 15 µg of soluble proteins from each sample were separated by SDS-polyacrylamide gel electrophoresis and blotted into a membrane. Immunodetection experiments using the anti-AtEm antibody showed the presence of the two expected bands in the wild-type seed extracts (Fig. 4). However, neither of these bands was detected in lec1-1 seed protein extract. The pre-immune sera did not react with any protein in the seed extracts. Because the amount of proteins loaded on the gel was the same for the two samples and the total protein content is much lower in lec1-1 than in wild-type seeds (Fig. 2
), these data indicate that neither AtEm1 nor AtEm6 proteins accumulate to a significant level in lec1-1 mature seeds. Moreover, bands of a similar or higher apparent mobility to AtEm1 protein were detected in the total protein extracts of lec1-1 seeds (Fig. 2), indicating that an extensive non-specific protein degradation is not the reason for the absence of detection of the AtEm proteins.
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Gene promoter activity in leafy cotyledon seeds
The AtEm1 promoter/ß-glucuronidase (AtEm1::GUS) reporter gene fusion (Hull et al., 1996) was introduced into the lec1-1, lec1-2 and fus3-2 mutant plants by conventional genetic crossing. The fus3-2 seeds were kindly provided by Dr Simon Miséra (IPK, Gatersleben). Heterozygous mutant plants were crossed with transgenic plants homozygous for the AtEm1::GUS construct. GUS activity was analysed in wild-type and mutant F2 dry seeds extracted from the same siliques of at least three independent crosses. Since both types of seeds develop within the same pod, varying physiological conditions can be excluded as an explanation for the differences in the GUS activity. As a control, the ABI3 gene promoter fused to the ß-glucuronidase gene (Parcy et al., 1994) was also introduced in lec1-1 and fus3-2 genetic backgrounds and similarly analysed. AtEm1 promoter activity was about 9-fold higher in lec1-1 and fus3-2 seeds compared to wild-type (Fig. 5). In contrast, a similar level of GUS activity was observed in lec1-2 seeds. These results with lec1-1 and lec1-2 seeds are consistent with the pattern of AtEm1 mRNA accumulation (Fig. 1). On the other hand, the levels of GUS activity directed by the ABI3 construct were not significantly affected in lec1-1 or fus3-2 seeds compared with wild-type.
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The time-course of AtEm1 and ABI3 promoter activity during seed development was determined in wild-type, lec1-1 and fus3-2 seeds by fluorometric assay (Fig. 6
). In wild-type AtEm1::GUS A. thaliana transgenic plants, GUS activity was detected only in the embryo during late stages of seed development (CM Vicient, unpublished data). At all the studied stages of development (mid-cotyledonary to mature seed) the level of expression of the AtEm1::GUS construct was higher in the lec1-1 and fus3-2 than in the wild-type seeds, whereas the level of expression of the ABI3::GUS construct was similar. As the lec1-1 embryos are not easily distinguishable from wild-type before the cotyledonary stage, it was not possible to perform fluorometric assays at earlier stages. However, at the cotyledonary stage the level of AtEm1 promoter activity in lec1-1 and fus3-2 seeds was several folds higher than in wild-type, suggesting that AtEm1 promoter began to be active earlier in seed development in the mutant embryos than in wild-type.
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Abscisic acid (ABA) induces the accumulation of the AtEm1 mRNA and the expression of the AtEm1 promoter in A. thaliana immature seeds (Gaubier et al., 1993
; Bies et al., 1998). ABA was analysed to discover whether it was also able to induce the AtEm1 promoter in lec1-1 immature seeds. Wild-type and lec1-1 immature AtEm1::GUS transgenic seeds extracted from the same siliques were incubated in the presence of 50 µM ABA. Wild-type and lec1-1 seeds were incubated in parallel in the absence of ABA as a control. After 24 h, the activity of the AtEm1 promoter in wild-type seeds was 40% higher in ABA-treated than in the untreated control seeds (Fig. 7). An increase was observed in lec1-1 seeds by the same order of magnitude (60%), indicating that the lec1 mutation does not alter ABA responsiveness.
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Deletion analysis of the putative cis-acting elements responsible for the high level of AtEm1 promoter activity in lec1-1 seeds
In order to identify the regions in the AtEm1 promoter involved in the regulation of the gene by the LEC1-dependent regulatory pathway, 5'-deletion constructs of the AtEm1 promoter were introduced into lec1-1 genetic background. Homozygous transgenic plants were crossed with lec1-1 heterozygous plants. The -1443 construct corresponds to the AtEm1 promoter construct used in previous experiments. All the plants of the different crosses were grown in parallel. GUS activity was determined in wild-type and lec1-1 F2 seeds as in previous experiments. GUS stimulation was defined as the relative increment of GUS activity in lec1-1 seeds compared to the wild type. The results obtained (Table 1) can be summarized in four points: (1) in the -1443 construct stimulation of GUS activity in the mutant was 10.6-fold, similar to the previously observed values (Fig. 5); (2) this stimulation in the GUS activity disappeared when the region -1443 to -197 was deleted; (3) 31.2 GUS stimulation was detected after the deletion of the next 15 bases (-197 to -182); and (4) GUS activity and GUS stimulation were reduced to very low levels after the deletion of the -182 to -119 region. These results suggest the existence of at least two regions in the AtEm1 promoter involved in the interaction with LEC1 regulatory pathway.
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Discussion |
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Leafy cotyledon1 mutants have been isolated on the basis of the morphological alterations in the embryo but, in addition to these alterations, the leafy cotyledon1 mutant embryos are also severely affected in several physiological aspects such as tolerance to desiccation, precocious germination and accumulation of storage lipids and proteins (Meinke, 1992
; Meinke et al., 1994; West et al., 1994). This complex phenotype suggests that the expression of many genes may be affected. Supporting this hypothesis, the mRNA steady-state level of all the lea genes tested was altered in the mutant seeds. Although the exact function of the protein products of many of the lea genes is not known, they may be involved in the acquisition of tolerance to desiccation (Delseny et al., 1994). Interestingly, leafy cotyledon1 seeds are intolerant to desiccation, so this phenotype could be correlated with an alteration in the expression of the genes conferring to the embryo the tolerance to desiccation. Accordingly, it has been shown that AtEm1 and AtEm6 proteins are present at an undetectable level in lec1-1 seeds. On the other hand, the mRNA accumulation of PAP85, an A. thaliana gene homologous to the broad bean vicilin gene, was reduced in lec1-1 and lec1-2 seeds. This is in agreement with the observation that mature protein bodies are not present in lec1 embryos (Meinke et al., 1994). Moreover, the accumulation of three cruciferin mRNAs is reduced by at least 50% in the lec1-1 mutant (Parcy et al., 1997), a 7S storage protein gene promoter is not operative in lec1-2 embryos (West et al., 1994) and the expression of cruciferin and 2S albumin genes is reduced in fus3-2 seeds (Bäumlein et al., 1994). A strong reduction was also observed in the accumulation of the storage proteins in total extracts of the mutant seeds.
LEC1, FUS3 and ABI3 proteins are key elements in the regulation of seed development. Accordingly to Meinke et al., LEC1 and FUS3 might act in the same regulatory pathway and separately from the ABI3 pathway (Meinke et al., 1994). Later, a study using double mutants of the three regulatory genes ABI3, lec1 and fus3 suggested that these three genes act in concert to regulate genes involved in seed maturation (Parcy et al., 1997). The results of this study support this scenario. lec1-1 and fus3-2 mutants have the same effect over the activity of the gene promoters tested, and the overall patterns of protein accumulation in fus3-2 and lec1 mature seeds are similar. These results are in agreement with the hypothesis that FUS3 and LEC1 are in the same regulatory pathway. On the other hand, the activity of ABI3 promoter is not significantly affected in the lec1-1 or fus3-2 seeds. Moreover, the activity of the AtEm1 gene promoter is ABA responsive in lec1-1 immature embryos and it is known that ABI3 is necessary for this response (Parcy et al., 1994). These data support the idea that ABI3 and LEC1/FUS3 proteins are in separate regulatory pathways. However, for example, AtEm6 or M10, require ABI3 and LEC1 for expression (Parcy et al., 1994), and the same was observed for the expression of two MYB genes (Kirik et al., 1998b). These results indicate that the ABI3 and LEC1 pathways interact in the regulation of at least some genes.
Increasing observations illustrate the complex regulation of the expression of the AtEm1 gene. In physiological conditions, AtEm1 is strongly expressed only during late embryogenesis. The expression of AtEm1 is reduced in the abi3 and aba1 mutants (Parcy et al., 1994). On the other hand, it has been shown that AtEm1 expression is increased in lec1-1 mutants, indicating that LEC1 protein may act as an inhibitor for AtEm1 expression. This over-expression behaviour of AtEm1 in lec1-1 background was further substantiated in two independent ways. First, Northern blot kinetics revealed that the gene accumulates much earlier in lec1-1 than in wild type. This observation is correlated with the fact that lec1 begins to be expressed at early stages of development (Lotan et al., 1998). Second an AtEm1::GUS fusion is also expressed earlier and to a higher level in the lec1-1 and fus3-2 genetic backgrounds. The activity of the ABI3 promoter is not affected in the same way. This is not the first example in which the expression of a gene is increased in the lec1-1 mutant, for instance, the expression of the AtMYB13 gene is also increased in lec1-1 embryos compared to wild type (Kirik et al., 1998a). Interestingly, as in the case of AtEm1, the transcript of AtMYB13 is not detected in the abi3 mutant. The ABA response of the AtEm1::GUS construct, which is mediated by ABI3 is not significantly affected by the lec1-1 mutation. This suggests that ABI3 and LEC1 control two distinct regulatory pathways and that AtEm1 is the target of both of them. A fus3-2 mutant as a recipient for the construction has been used as an independent control. It is interesting that the AtEm1 promoter is over-expressed as well in the fus3-2 background, suggesting that FUS3 might also repress AtEm1. The exact nature of the interaction between LEC1 and AtEm1 promoter is not known. The 5' promoter deletion analysis showed that, at least, two regions in the promoter are implicated in the regulation of the AtEm1 gene by the LEC1-dependent regulatory pathway.
The increased expression observed in lec1-1 was not observed in lec1-2. Some other differences in gene expression and protein accumulation have been reported between both alleles. For example, differences in the accumulation of AGL15 (AGAMOUS-like MADS domain) protein have been reported (Perry et al., 1996) and the expression of the PAP51 gene is also differentially affected by the two allelic mutations. Although lec1-1 and lec1-2 alleles show very similar phenotypes, general morphology, internal anatomy and response in culture, it is likely that lec1-1 could correspond to a null allele whereas it is conceivable that lec1-2 might be leaky.
These results show that although AtEm1 mRNA accumulates to high levels in lec1-1 the protein itself does not accumulate. These data suggest the existence of an additional post-transcriptional regulation. Some previous data also support this hypothesis. It is possible to induce the accumulation of AtEm1 mRNA in response to ABA in immature seeds (Gaubier et al., 1993) and in leaves of transgenic plants ectopically expressing ABI3 (Parcy et al., 1994), however, in both cases, the protein does not accumulate (Bies et al., 1998). It remains unclear whether the protein is not translated, is degraded or both. In the second case, the degradation must be something more or less specific because proteins of relative high molecular weights are still present in protein extracts of lec1 seeds. The existence of specific proteases degrading Em protein has been reported in wheat (Taylor and Cuming, 1993a, b) and there is evidence of its existence in A. thaliana(Bies et al., 1998).
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Acknowledgments |
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We thank J Giraudat and F Parcy for the gift of the ABI3::GUS transgenic seeds and for fruitful and stimulating discussions. We are grateful for the gift of the AtEm1::GUS transgenic seeds from G Hull, and for the gift of mutant seeds from D Meinke, S Miséra and J Harada. We are grateful to AH Schulman, R Cooke, S Prat and D Ludevid for their help in preparing this manuscript. CMV was the recipient of a postdoctoral fellowship from the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). This work was supported by the CNRS and by the EC BIOTECH Program (Grant No. BIO4-CT96-0062), and benefits from the joint CNRS-CSIC Laboratoire Européen Associé Perpignan-Barcelone for Plant Molecular and Cellular Biology.
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Notes |
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1 Present address and to whom correspondence should be sent. Institute of Biotechnology, University of Helsinki, Biocenter 1, PO Box 56, Viikinkaari 9, FIN-00014 Helsinki, Finland. Fax: +358 9 708 59570. E-mail:carlos.vicient{at}helsinki.fi
2 Present address: Department of Biology, McDonnell Hall Rm248, Washington University, St Louis, MO 63130, USA.
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References |
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Baker J, Steele C, Dure III L.1988. Sequence and characterisation of 6 Lea proteins and their genes from cotton. Plant Molecular Biology 11, 277–291.[Web of Science]
Bassüner R, Van Hai N, Jung R, Saalbach G, Müntz K.1987. The primary structure of the predominating vicilin storage protein subunit from field bean seeds (Vicia faba L. var. Minor cv. Fribo). Nucleic Acids Research 15, 9609.
Bäumlein H, Miséra S, Luerßen H, Kölle K, Horstmann C, Wobus U, Müller AJ.1994. The FUS3 gene of Arabidopsis thaliana is a regulator of gene expression during late embryogenesis. The Plant Journal 6, 379–387.[Web of Science]
Bies N, Aspart L, Carles C, Gallois P, Delseny M.1998. Accumulation and degradation of Em proteins in Arabidopsis thaliana: evidence for post-transcriptional controls. Journal of Experimental Botany 49, 1925–1933.
Bradford MM.1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[Web of Science][Medline]
Cohen A, Bray EA.1992. Nucleotide sequence of an ABA-induced tomato gene that is expressed in wilted vegetative organs and developing seeds. Plant Molecular Biology 18, 411–413.[Web of Science][Medline]
Cooke R, Raynal M, Laudié M, et al.1996. Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs. The Plant Journal 9, 101–124.[Web of Science][Medline]
de Vries SC.1998. Making embryos in plants. Trends in Plant Science 3, 451–452.
Delseny M, Gaubier P, Hull G, Saez-Vazquez J, Gallois P, Raynal M, Cooke R, Grellet F.1994. Nuclear genes expressed during seed desiccation: relationship with responses to stress. In: Basra AS, ed. Stress-induced gene expression. Reading: Harwood Academic Publishers, 25–59.
Devic M, Hecht V, Berger C, Lindsey K, Delseny K, Gallois P.1995. Assessment of promoter trap as a tool to study zygotic embryogenesis in Arabidopsis thaliana. Comptes Rendus de l'Académie des Sciences—Series III—Sciences de la vie 316, 1194–1199.
Farrell Jr RE.1993. Electrophoresis of RNA. In: Farrell Jr RE, ed. RNA methodologies. A laboratory guide for isolation and characterization. San Diego: Academic Press, 125–157.
Finkelstein RR.1993. Abscisic acid-insensitive mutations provide evidence for stage-specific signal pathways regulating expression of an Arabidopsis late embryogenesis-abundant (lea) gene. Molecular and General Genetics 238, 401–408.
Gaubier P, Raynal M, Hull G, Huestis GM, Grellet F, Arenas C, Pages C, Delseny M.1993. Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation. Molecular and General Genetics 238, 409–418.
Grellet F, Delcasso-Tremouysaygue D, Delseny M.1989. Isolation and characterization of an unusual repeated sequence from the ribosomal intergenic spacer of the crucifer Sisymbrium irio. Plant Molecular Biology 12, 695–706.
Harada JJ, DeLisle AJ, Baden CS, Crouch ML.1989. Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development. Plant Molecular Biology 12, 395–401.
Harada JJ, Lotan T, Fischer RL, Goldberg RB.1998. ...Response: embryos without sex. Trends in Plant Science 3, 452–453.
Hull GA, Bies N, Twell D, Delseny M.1996. Analysis of the promoter of an abscisic acid responsive late embryogenesis abundant gene of Arabidopsis thaliana. Plant Science 114, 181–192.
Keith K, Kraml M, Dengler NG, McCourt P.1994. fusca3: a heterochromic mutation affecting late embryo development in Arabidopsis. The Plant Cell 6, 589–600.
Kirik V, Kölle K, Balzer HJ, Bäumlein H.1996. Two new oleosin isoforms with altered expression in seeds of Arabidopsis mutant fus3. Plant Molecular Biology 31, 413–417.[Web of Science][Medline]
Kirik V, Kölle K, Wohlfarth T, Miséra S, Bäumlein H.1998a. Ectopic expression of a novel MYB gene modifies the architecture of the Arabidopsis inflorescence. The Plant Journal 13, 729–742.[Web of Science][Medline]
Kirik V, Kölle K, Miséra S, Bäumlein H.1998b. Two novel MYB homologues with changed expression in late embryogenesis-defective Arabidopsis mutants. Plant Molecular Biology 37, 819–827.[Web of Science][Medline]
Leung J, Giraudat J.1998. Abscisic acid signal transduction. Annual Review of Plant Physiology and Plant Molecular Biology 49, 199–222.[Web of Science]
Lotan T, Ohto M, Yee KM, West MAL, Lo R, Kwong RW, Tamagishi K, Fisher RL, Goldberg RB, Harada JJ.1998. Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93, 1195–1205.[Web of Science][Medline]
Luerßen H, Kirik V, Herrmann P, Miséra S.1998. FUSCA3 encodes a protein with a conserved Vp1/ABI3-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana. The Plant Journal 15, 755–764.[Web of Science][Medline]
Meinke D.1992. A homeotic mutant of Arabidopsis thaliana with leafy cotyledons. Science 258, 1647–1650.
Meinke DW, Franzmann LH, Nickle TC, Yeung EC.1994. Leafy Cotyledon mutants of Arabidopsis. The Plant Cell 6, 1049–1064.[Abstract]
Miséra S, Müller AJ, Weiland-Heidecker U, Jürgens G.1994. The FUSCA genes of Arabidopsis: negative regulators of light responses. Molecular and General Genetics 244, 242–252.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473–497.
Ogata F, Miyata T, Fujii N, Yoshida N, Noda K, Makisumi S, Ito A.1991. Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus. Journal of Biological Chemistry 266, 16715–16721.
Parcy F. Valon C, Kohara A, Miséra S, Giraudat J.1997. The ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY COTYLEDON1 loci act in concert to control multiple aspects of Arabidopsis seed development. The Plant Cell 9, 1265–1277.[Abstract]
Parcy F, Valon C, Raynal M, Gaubier-Comella P, Delseny M, Giraudat J.1994. Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3 locus and of endogenous abscisic acid. The Plant Cell 6, 1567–1582.[Abstract]
Perry SE, Nichols KW, Fernandez DW.1996. The MADS domain protein AGL15 localizes to the nucleus during early stages of seed development. The Plant Cell 8, 1977–1989.[Abstract]
Raynal M, Depigny D, Cooke R, Delseny M.1989. Characterization of a radish nuclear genes expressed during late seed maturation. Plant Physiology 91, 829–836.
Raynal M, Guilleminot J, Gueguen C, Cooke R, Delseny M, Gruber V.1999. Structure, organization and expression of two closely related novel lea (late-embryogenesis-abundant) genes in Arabidopsis thaliana. Plant Molecular Biology 40, 153–165.[Web of Science][Medline]
Sambrook J, Fritsh EF, Maniatis T.1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.
Taylor RM, Cuming AC.1993a. Selective proteolysis of the wheat Em polypeptide. FEBS Letters 331, 71–75.[Web of Science][Medline]
Taylor RM, Cuming AC.1993b. Purification of an endoproteinase that digests the wheat ‘Em’ protein in vitro, and determination of its cleavage sites. FEBS Letters 331, 76–80.[Web of Science][Medline]
Vicient CM, Delseny M.1999. Isolation of total RNA from Arabidopsis thaliana seeds. Analytical Biochemistry 268, 412–413.[Web of Science][Medline]
West MAL, Yee KM, Danao J, Zimmerman JL, Fischer RL, Goldberg RB, Harada JJ.1994. LEAFY COTYLEDON1 is an essential regulator of late embryogenesis and cotyledon identity in Arabidopsis. The Plant Cell 6, 1731–1745.
Wobus U, Weber H.1999. Seed maturation: genetic programmes and control signals. Current Opinion in Plant Biology 2, 33–38.[Web of Science][Medline]
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