Journal of Experimental Botany, Vol. 51, No. 348, pp. 1211-1220,
July 2000
© 2000 Oxford University Press
Original papers |
Differential expression of the Arabidopsis genes coding for Em-like proteins1
Laboratoire de Physiologie et Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique UMR 5545, Université de Perpignan, 66860 Perpignan Cedex, France
Received 3 September 1999; Accepted 7 March 2000
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Abstract |
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Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the ß-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).
Key words: Embryogenesis, LEA, promoter, transgenic.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Seed maturation is characterized by a desiccation process. During desiccation, a number of specific proteins referred to as LEA (Late Embryogenesis Abundant proteins) accumulate in the embryo (Baker et al., 1988
; Hughes and Galau, 1989). According to their accumulation pattern and physico-chemical proprieties, it has been suggested that LEA proteins could be involved in seed desiccation tolerance (Dure et al., 1989; Hughes and Galau, 1989; Dure 1993; Leprince et al., 1993; Xu et al., 1996; Kermode, 1997). LEA proteins have a widespread distribution among plant species. Five major groups of LEA proteins have been described on the basis of their amino acid sequence homologies (Dure et al., 1989; Delseny et al., 1993). Expression of many lea genes can be precociously induced in immature seeds or in vegetative tissues upon ABA treatment or by osmotic or water-deficit stress (Skriver and Mundy, 1990; Jakobsen et al., 1994).
The first LEA protein was identified in wheat, and corresponded to the so-called Em protein (Cuming and Lane, 1979). Since then, several Em-like coding genes have been isolated and characterized in many monocot and dicot species (Vicient et al., 1998), all of them encoding proteins having the highly conserved hydrophilic 20 amino acid motif (Dure et al., 1989). In some species, the Em genes are encoded by multigene families, sometimes encoding proteins with different numbers of the 20 amino acid motif arranged in tandem, as for example in cotton (Galau et al., 1992), maize (Williams and Tsang, 1992), barley (Espelund et al., 1992; Stacy et al., 1995), Arabidopsis (Gaubier et al., 1993), mung bean (Manickam et al., 1996) or soybean (Calvo et al., 1997). Em genes are physiologically expressed only during the later steps of seed maturation (Hughes and Galau, 1989, 1991; Raynal et al., 1989; Almoguera and Jordano, 1992; Espelund et al., 1992; Gaubier et al., 1993; Hollung et al., 1994; Parcy et al., 1994) and are not inducible in adult vegetative tissues. However, the expression of these genes can be induced by ABA application in immature seeds and young seedlings (Williamson and Quatrano, 1988; Morris et al., 1990, Williams and Tsang, 1991; Almoguera and Jordano, 1992; Espelund et al., 1992; Gaubier et al., 1993; Hollung et al., 1994; Manickam et al., 1996). Their expression is reduced in ABA-deficient mutants and suppressed in some ABA-insensitive mutants (McCarty et al., 1991; Butler and Cuming, 1993; Parcy et al., 1994; Vasil et al., 1995). There are few examples in which the expression of the various members of an Em gene family has been characterized. The members of the barley B19 (Em-like) gene family accumulate to different levels in the developing embryo and respond differentially to salt-stress (Espelund et al., 1992).
Despite this information on Em gene expression, very little information is known about the spatial patterns of expression of the Em genes in any species. In situ hybridization using a carrot EMB-1 probe showed that most of this mRNA is localized in the meristematic regions, particularly in the procambium (Wurtele et al., 1993). GUS activity in transgenic tobacco seeds transformed with the promoter of the wheat Em gene fused to the uidA reporter gene showed uniform GUS activity throughout the embryo (Marcotte et al., 1989). The Arabidopsis AtEm1 promoter directs GUS activity uniformly throughout the embryo of transgenic tobacco (Hull et al., 1996).
During recent years, this laboratory has focused on the analysis of expression of Em genes in Arabidopsis thaliana. The genome of Arabidopsis thaliana contains two Em genes, AtEm1 and AtEm6 (Gaubier et al., 1993). The AtEm6 protein has one copy of the 20-amino-acid motif, whereas AtEm1 has four copies arranged in tandem. Although the AtEm1 and AtEm6 mRNAs accumulate to high levels in dry seeds, they appear to be differentially regulated. AtEm1 is expressed less abundantly and at a slightly earlier stage in seed development than AtEm6 mRNA (Gaubier et al., 1993; Bies et al., 1998) and AtEm1 and AtEm6 are differentially regulated by the ABI3 protein (Parcy et al., 1994). The promoter sequences of these genes do not reveal any similarity (Gaubier et al., 1993). As a part of the efforts to understand the differential regulation of the two Em genes in Arabidopsis thaliana, the spatial patterns of expression of both genes have been studied. In situ RNA hybridization and transgenic Arabidopsis plants containing the promoters of these genes fused to the ß-glucuronidase reporter gene were used. This study shows that AtEm1 and AtEm6 genes are subjected to different tissue-specific control and that the AtEm1 promoter, but not AtEm6, drives GUS activity in pollen and collapsed nectaries.
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Materials and methods |
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In situ hybridization
Mature Arabidopsis seeds were fixed in 4% paraformaldehyde, 0.25% glutaraldehyde, 0.1 M NaCl, 10 mM sodium phosphate pH 7.2. Seeds were embedded in 1% agarose in sodium phosphate buffer and washed twice in the same buffer. Fixed samples were dehydrated by a passage through ethanol/water-solutions. The dehydrated samples were then incubated with xylene. The seeds were embedded in paraffin at 60 °C for 2 d replacing paraffin four times. The embedded material was cut into 7 µm thick slices with a rotatory microtome and placed onto poly-D-lysine coated slides. The RNA probes were prepared using pBluescriptII constructs (Stratagene) and the T7 or T3 RNA polymerases in the presence of digoxygenine-uridine 5' triphosphate (Boehringer). Final probe concentration was 100 ng ml-1 in hybridization buffer (0.3 M NaCl, 50% formamide, 1xDenhardt's solution, 10% dextran sulphate, 10 mM TRIS-HCl pH 7.5, 1 mM EDTA, 60 mM DTT, 150 µg ml-1 yeast tRNA, and 500 µg ml-1 poly(A) RNA). Labelled probes were heated at 80 °C for 5 min and added to the hybridization buffer. Hybridization controls with sense probes were systematically carried out. Prior to hybridization, paraffin was removed and sections rehydrated by passage through ethanol/water solutions containing increasing amounts of water. The sections were subjected to proteinase K digestion in 0.5 M NaCl, 100 mM TRIS-HCl pH 7.5, 1 mM EDTA at 37 °C for 30 min. Samples then were incubated in 0.2% glycine in PBS buffer (10 mM sodium phosphate pH 7.5, 150 mM NaCl), rinsed in PBS, treated with 0.5% acetic anhydride in 0.1 M triethanolamide pH 8.0 for 10 min., rinsed again in PBS and then in 2xSSC. Hybridization was carried out at 42 °C overnight in a humid chamber. After hybridization, the samples were washed with 2xSSC at 37 °C. Digestion of non-specifically bound probe was done with 25 µg ml-1 RNAse in 0.5 M NaCl, 100 mM TRIS-HCl pH 7.5, EDTA 1 mM at 37 °C for 30 min, and then washed three times in the same buffer and conditions. After additional washes (once for 10 min, 2xSSC at 55 °C, twice for 20 min, 0.2xSSC at room temperature and once for 5 min in PBS), sections were blocked for 1 h at room temperature with 1% BSA, 0.5% blocking reagent (Boehringer) in TTBS (0.15% Triton X100, 150 mM NaCl, 100 mM TRIS-HCl pH 7.5), and then incubated for 1 h at room temperature with antidigoxygenin antibodies coupled with alkaline phosphatase in blocking buffer. Unbound antibody was removed by several 5 min washes in TTBS. Alkaline phosphatase activity was revealed with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium. Sections were examined in a differential interference contrast optic microscope (Zeiss axioplan).
DNA constructs and transformation
The construction pEm1 containing the promoter region of the AtEm1 gene is described elsewhere (pEm1.1443 in Hull et al., 1996). pEm6 contains the entire cloned promoter region of AtEm6 gene (Gaubier et al., 1993). A fragment extending from -1188 bp to +85 bp (relative to start of transcription) was excised by digestion with SphI and AhaII, and ligated into pBI101.2 digested with Sph and AccI. AhaII cuts 1 bp 3' of the initiation codon of AtEm6 and ligation into the AccI site of pBI.101.2 generates a transcriptional fusion with a 13 amino acid N-terminal extension. The sequences of the GUS fusion junctions were checked by sequencing.
pEm1 and pEm6 constructs were transferred to Agrobacterium tumefaciens LBA4404 by triparental mating. Roots of Arabidopsis thaliana cv. C24 were transformed as described previously (Clarke et al., 1992). Transformants were transferred to soil and grown in a growth room at 25 °C, 16 h day. Most analyses were performed with plants of three lines per construct homozygous for the T-DNA insertion with medium to high levels of GUS activity in seeds. All transgenic plants were grown according to the French safety guidelines and the French Genetic Engineering Commission approved experiments with these plants.
Fluorometric measurement and histochemical localization of ß-glucuronidase (GUS) activity
GUS assays were carried out essentially as described previously (Jefferson, 1987). Fluorometric GUS assays based on the enzymatic conversion of 4-methylumbelliferyl glucuronide (4-MUG) to 4-methylumbelliferone (4-MU) were carried out in microtitre plates using a Fluoroskan II automated fluorometer (Labsystems, Helsinki) in conjunction with computer software DeltasoftTM (BioMetallics Inc. Princetown). Plant samples for GUS assays were ground up in 50 mM TRIS-HCl pH 7.0, 0.1% Triton, 10 mM EDTA, 3 mM DTT. Protein content of the extracts was determined according to Bradford (Bradford, 1976). Histochemical staining of GUS activity was carried out as described previously (Hull and Devic, 1995) using X-GLUC (5-bromo-4-chloro-3-indolyl-ß-D-glucuronide) as substrate. Transverse sections of stained tissues were prepared using cleared and dehydrated tissues embedded in HistoresinTM (Leica Instruments GmbH) according to manufacturer's protocols. 10 micrometer sections were made with a microtome and mounted on a glass slide.
Abscisic acid treatments
For ABA treatment, (±)-ABA (Sigma) was added to water from a stock solution. The stock solution consisted in ABA dissolved in ethanol to a final concentration of 50 mM. Seeds were germinated and at 7 d or 21 d after germination sprayed with a 100 µM ABA. Control plants were sprayed with water supplemented with the equivalent concentration of ethanol. Both groups of plants were grown alongside each other in a growth chamber (25 °C, 16 h day) for 36 h.
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Results |
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Localization of the expression of AtEm1 and AtEm6 mRNAs in the embryo of Arabidopsis thaliana
RNA slot blot analysis showed that the Arabidopsis Em genes are strongly expressed in the mature seed (Gaubier et al., 1993
). To compare the distribution of AtEm1 and AtEm6 transcripts in the mature embryo, mRNA in situ hybridizations were performed. Dry Arabidopsis seeds were embedded in paraffin, sectioned, and hybridized with antisense digoxygenin-labelled RNA probes specific for each of the Arabidopsis Em genes. These probes correspond to the 3' untranslated region of the genes and control experiments demonstrated that there was no cross-hybridization (Gaubier et al., 1993). A different spatial pattern of hybridization was observed using AtEm1 or AtEm6 probes. AtEm1 is preferentially expressed in the provascular tissue of the embryonic axis and in the root tip (Fig. 1A). Slight hybridization signals were also detected in the shoot apical meristem and in the cotyledon provascular bundles. The specificity of the hybridization was confirmed by the lack of signal in the hybridization controls using sense RNA probes that had been labelled to the same specific activity as the antisense ones (Fig. 1B). The AtEm6 antisense probe (Fig. 1C) hybridized throughout the entire embryo with a stronger signal in provascular tissues and shoot apical meristem. As in the case of the AtEm1 gene, the hybridization controls using the sense RNA probe labelled to the same specific activity as the antisense one, no hybridization signal was detected (data not shown). AtEm1 and AtEm6 transcripts did not accumulate in the seed coat.
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AtEm1 and AtEm6 promoter-driven expression in seeds of transgenic Arabidopsis
The putative full-length promoters of AtEm1 and AtEm6 genes were fused to the uidA reporter gene (pEm1 and pEm6 constructs) and introduced into Arabidopsis thaliana. Eight independently transformed transgenic plants carrying pEm1 and 28 carrying pEm6 were generated. GUS activity was assayed fluorimetrically in mature seeds of the T0 plants (Fig. 2). 50 seeds were pooled and assayed for each of the independently transformed transgenic plants. A wide range of GUS activity was observed among the plants transformed with the same construct, which is not unusual for promoter analysis using the uidA reporter gene (Peach and Velten, 1991). A similar variation was previously observed for the AtEm1 promoter in transgenic tobacco (Hull et al., 1996). Despite the interclonal variation, the average GUS activity in seeds of transgenic plants transformed with pEm6 was 4.6-fold higher (39.6 nmol 4-MU min-1 mg-1 protein) than in seeds transformed with pEm1 (8.7). Seeds of T0 transgenic plants were collected and plated on selective medium. Lines homozygous for the T-DNA insertion were initiated from T2 plants that gave 100% resistant seedlings after selfing. Three independent insertions per construct with medium to high levels of GUS activity in seeds were fixed in this way and used for further analysis (Fig. 2). The homozygosity for the T-DNA insertion was confirmed by the 100% of GUS positive seeds in histochemical assays. In all of the histochemical and fluorometric GUS analyses all three lines transformed with the same construct presented a similar pattern of promoter activity.
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The developmental regulation of the AtEm1 and AtEm6 promoters during the course of embryogenesis was analysed. Seeds of transgenic Arabidopsis were sampled at intervals of 2 d and assayed for GUS activity (Fig. 3
). In the lines transformed with pEm1 the promoter activity remained null or very low in seeds before 11 d after pollination (d.a.p.), increased until 17 d.a.p. and then decreased in mature seeds. The time-course of GUS induction in plants transformed with pEm6 was slightly different. The AtEm6 promoter remained inactive in immature transgenic seeds until about 13 d.a.p., then GUS activity increased rapidly until a maximum level at 19 d.a.p. and finally decreased a bit in mature seeds.
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The spatial patterns of expression were examined by quantitative analysis of GUS activity in various organs of the plant (Fig. 4
). Both promoters directed the highest GUS activities in mature seeds. Vegetative tissues (leaves, caulinar leaves, stems, and roots) did not show GUS expression in any of the transgenic plants. The AtEm1 promoter also directed uidA expression in flowers. On the contrary, the pEm6 construct did not show activity in flowers. GUS activity was also analysed in transgenic plants different times after germination. A rapid decrease in the GUS activity was observed after germination in both groups of transgenic plants.
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It was tested whether AtEm1 and AtEm6 promoters were ABA-inducible in transgenic Arabidopsis seedlings (Fig. 5
). Seeds were germinated and at 7 d after germination were sprayed with 100 µM ABA. Control plants were sprayed with water plus the equivalent amount of ethanol. Both groups of plants were grown alongside each other for 36 h. Application of exogenous ABA induced, on average, a 1.41-fold higher GUS activity in the seedlings transformed with pEm1 and 4.49-fold higher GUS activity in the seedlings transformed with pEm6.
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Histochemical staining of GUS activity was used for a higher resolution determination of tissue-specificity in Em expression. These analyses were done with the three transgenic lines selected per construct with similar results. Although the level of GUS activity varied from one line to another, the overall pattern of GUS activity was qualitatively the same in all the plants transformed with the same construct. Seeds of plants transformed with pEm1 or pEm6 were dissected and the embryos were assayed for GUS activity. In embryos from plants transformed with pEm1, GUS activity was localized predominantly in the provascular tissues and in the root tip and apical meristem (Fig. 6A
). In embryos from plants transformed with pEm6, GUS activity was distributed uniformly throughout the embryo (Fig. 6B). The seed coat did not show GUS activity in any of the lines. The spatial patterns of GUS activity in seedlings upon ABA treatment were determined using 21 d.a.g. seedlings and the previously used treatment conditions. Untreated 21 d.a.g. seedlings did not show X-GLUC staining (data not shown). After ABA treatment, GUS activity was detected in the roots of either seedlings transformed with pEm1 or with pEm6 (Fig. 6C, D). Transverse sections of the ABA treated roots showed that AtEm1 promoter activity was located in the central cylinder (vascular cells and pericycle) (Fig. 6E) whereas the activity of the AtEm6 promoter was located in the external tissues of the root (cortex, epidermis and root hairs) (Fig. 6F). Staining was also detected after ABA treatment in the base of the first true leaves in plants transformed with pEm1 (Fig. 6G), but in plants transformed with pEm6 the staining was located in the base of the cotyledons (Fig. 6H). Histochemical staining of detached flowers of plants transformed with the pEm1 construct showed that GUS activity was localized in the anthers (Fig. 6I). A closer view demonstrated that anthers-walls were not stained (Fig. 6J) and that all the GUS activity was located exclusively in the pollen grains (Fig. 6K). This GUS activity was still visible in the stigma of the fertilized flowers due to the attached pollen grains (Fig. 6L). In plants transformed with the pEm1 construct, GUS activity was also detected in the collapsed nectaries at early stages of silique development (less than 1 cm long) (Fig. 6M).
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In order to determine whether GUS expression in pollen grains was under developmental control, flowers of the same inflorescence at different stages of development were assayed fluorometrically for GUS activity (Fig. 7
). No GUS activity was detected in floral buds, but high GUS expression was detected in the flowers containing mature pollen grains with higher activities during anthesis. GUS activity decreased after pollination.
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Discussion |
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The expression patterns of the two Arabidopsis Em-like genes (AtEm1 and AtEm6) have been studied using in situ hybridization and Arabidopsis transgenic plants containing the promoters of these genes fused to the uidA reporter gene. The spatial pattern of promoter activity observed in the embryos were very similar to the patterns of mRNA accumulation obtained. Moreover, the patterns of promoter activity during seed development were similar to the patterns of mRNA accumulation previously observed (Gaubier et al., 1993
; Parcy et al., 1994). These data suggest that the promoter fragments used in the pEm1 and pEm6 constructs contain the specific cis elements required for their correct regulation in Arabidopsis.
The experiments presented here demonstrate that the expression of the two Em genes of Arabidopsis is subject to differential, tissue-specific control. Although the expression of both genes is highest in the mature embryo (Gaubier et al., 1993), AtEm1 mRNA accumulates mainly in the provascular tissues of the embryo axis and in meristems, while AtEm6 mRNA is distributed throughout the embryo. Previous data, based on RNA blot analyses, indicated that AtEm1 mRNA accumulates at a lower level in seeds than does AtEm6 mRNA (Gaubier et al., 1993). This observation is probably due to their different spatial patterns of expression, since AtEm1 is expressed mainly in vascular tissues and meristems, and these tissues represent only a small portion of the whole seed. A differential pattern of promoter activity was also detected in the roots of ABA-treated young seedlings: AtEm1 promoter is active in the vascular tissues whereas AtEm6 is active in the external tissues.
AtEm1 and AtEm6 promoter activities disappeared rapidly after germination. A rapid disappearance of the AtEm1 and AtEm6 transcripts has also been observed during the first steps of germination (Bies et al., 1998). A similar situation has been observed in several lea genes (Hughes and Galau, 1989; Raynal et al., 1989; Goday et al., 1988; Harada et al., 1989; Espelund et al., 1992). However, the accumulation of some lea mRNAs can be re-induced in seedlings by treatment with ABA (Berge et al., 1989; Vilardell et al., 1991; Hong et al., 1992; Finkelstein, 1993). AtEm1 and AtEm6 promoters are also ABA inducible in seedlings. The presence of some putative ABA regulatory elements in the promoters of the AtEm1 and AtEm6 genes has been reported previously (Gaubier et al., 1993; Hull et al., 1996). In addition to these elements, a sequence (tCTCCGTaa) similar to the binding site of the VSF1 protein (GCTCCGTTG) located at position -200 in the promoter of the French bean (Phaseolus vulgaris L.) grp1.8 gene (Ringli and Keller, 1998) has been found in the AtEm1 promoter, at position -139 bp,. This fragment is sufficient and necessary to confer vascular specific expression to a minimal promoter. Interestingly, preliminary results indicate that the first 182 bp of the AtEm1 promoter are sufficient to direct GUS activity in provascular tissues of the embryo (G Hull, unpublished data). Further analysis will be necessary to determine which promoter sequences are involved in the vascular specific expression of AtEm1.
The pattern of AtEm1 promoter activity has been previously determined in transgenic tobacco (Nicotiana tabacum L.) (Hull et al., 1996). In this specie, the AtEm1 promoter directed the same organ and development specific activity as in Arabidopsis , with higher expression in mature embryos and some expression in pollen grains. However, the predominantly vascular expression of the AtEm1 promoter in the embryos of Arabidopsis was not observed in tobacco (Hull et al., 1996). These results suggest that at least some of the transcription factors required for the vascular expression of AtEm1 in Arabidopsis may be absent from tobacco, or transcription factors which recognize elements in the promoter of AtEm1 may be expressed more generally in the embryos of tobacco.The AtEm1 promoter-directed vascular predominant expression is present in the embryos of Brassica napus L. (C Vicient, unpublished data) indicating that the vascular regulatory factors are conserved in this species. Phylogenetically, B. napus is very much more closely related to Arabidopsis than tobacco.
The AtEm1 promoter is also active in pollen grains. GUS artefacts associated with the histochemical GUS activity assay in pollen have been reported (Mascarenhas and Hamilton, 1992). Uknes et al. have proposed that GUS expression in pollen may be due more to the coding region of the uidA gene than to the fused promoter (Uknes et al., 1993). However, the pEm1 and pEm6 constructs contain the same uidA gene coding region but only plants transformed with pEm1 show GUS activity in pollen grains. It is interesting that high levels of GUS activity were also driven in pollen by the promoters of some other ABA- or dehydration-inducible genes such as Tas14 (Parra et al., 1996), CDeT6–19 (Michel et al., 1994), CDeT27–45 (Michel et al., 1993), cor15a (Baker et al., 1994), kin1, cor6.6 (Wang and Cutler, 1995), osmotin (Kononowicz et al., 1992), and Xero2/lti30 (Rouse et al., 1996). The pEm1 construct also showed expression in pollen grains when it was introduced in tobacco (Hull et al., 1996) and B. napus (C Vicient, unpublished results). Pollen grains are highly desiccated tissues (Heslop-Harrison, 1987), suggesting that the expression of the AtEm1 promoter in these tissues could be due to desiccation. Interestingly, collapsed nectaries, in which the AtEm1 promoter is also active, are also highly desiccated tissues.
Although the exact function of the Em proteins is not known, they may be involved in seed desiccation tolerance (Dure et al., 1989; Hughes and Galau, 1989; Dure 1993; Leprince et al., 1993; Xu et al., 1996; Kermode, 1997). The expression of the Em protein in yeast cells attenuates the growth inhibition normally observed in media of high osmolarity and mitigates the detrimental effect of low water potential in a relatively non-specific manner (Swire-Clark and Marcotte, 1999). The variable number of hydrophilic repeats in the members of the Arabidopsis Em gene family, their differential mRNA abundance in dry seeds, their different temporal regulation during seed development and their distinct spatial distribution in the embryo may indicate that the two Em proteins could carry out at least slightly different functions, as has been suggested for the barley Em proteins (Espelund et al., 1992).
It can be concluded that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA). The exact nature of the factors determining this regulation and the functional implications of the different patterns of expression of the two genes remain to be elucidated.
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Acknowledgments |
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We are grateful to Dr Alan H Schulman for his help in preparing this manuscript. CMV was 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 N0. 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, Viikinkaari 6, PO Box 56, FIN-00014 Helsinki, Finland. Fax: +358 9 191 58 952. E-mail: carlos.vicient{at}helsinki.fi
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Abbreviations |
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ABA, abscisic acid; d.a.g., days after germination; d.a.p., days after pollination; GUS, ß-glucuronidase reporter gene; LEA, late embryogenesis abundant; 4-MU, 4-methylumbelliferone..
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