Journal of Experimental Botany, Vol. 52, No. 360, pp. 1587-1591,
July 1, 2001
© 2001 Oxford University Press
Short Communications |
The Arabidopsis AtEm1 promoter is active in Brassica napus L. and is temporally and spatially regulated
1 Génome et Developpement des Plantes, UMR 5096, CNRS, Universite de Perpignan, 52 avenue de Villeneuve, 66860 Perpignan Cedex, France
2 MERISTEM Therapeutics, 8 rue des Frères Lumière, 63100 Clemont-Ferrand, France
Received 14 September 2000; Accepted 2 March 2001
Abstract
The promoter of the Arabidopsis thaliana L. AtEm1 gene encoding a late embryogenesis abundant protein was fused to the ß-glucuronidase reporter gene and introduced into Brassica napus. The promoter is highly active in the vascular tissues of embryo and pollen grains and also active in petals, sepals, caulinar leaves, and carpels.
Key words: LEA protein, Brassica napus, promoter, AtEm1 gene.
Introduction
Late Embryogenesis Abundant proteins (LEA) are a group of hydrophilic proteins abundantly accumulated during seed desiccation and in mature embryos, and that rapidly disappear after germination (Delseny et al., 1993
). The expression of the lea genes can be induced in immature embryos by abscisic acid (ABA) and osmotic- or salt-stress treatment (Skriver and Mundy, 1990). These characteristics of expression and the physical properties of the proteins suggest that they may be involved in desiccation tolerance during seed development (Dure, 1993). Accordingly, the expression of Em proteins in yeast cells mitigates the detrimental effect of low water potentials (<Swire-Clark and Marcotte, 1999).
AtEm1 gene from Arabidopsis thaliana encodes for a group I LEA protein (Gaubier et al., 1993). AtEm1 mRNA accumulates in immature seeds and young seedlings in response to ABA. In Arabidopsis, AtEm1 mRNA specially accumulates in the vascular tissues of the embryo and its promoter has a similar pattern of expression (Vicient et al., 2000). However, when the same promoter was assayed in tobacco (Nicotiana tabacum L.) transgenic plants its activity was distributed uniformly throughout the embryo (Hull et al., 1996).
The spatial pattern of expression of the AtEm1 gene in embryos was particularly interesting for biotechnological purposes such as heterologous gene expression during late embryogenesis, so it was decided to study its expression in rapeseed (Brassica napus). Arabidopsis and Brassica species are phylogenetically very closely related and the sequence similarities between homologous genes is high (Arondel et al., 1992). For example, the coding regions of the AtEm6 Arabidopsis gene and its homologue in Brassica napus (BnEm6) are 79% similar at the nucleotide level (Vicient et al., 1998). This very close phylogenetic relationship allows, in principle, A. thaliana genes to be used directly in B. napus in a near homologous system.
Materials and methods
DNA constructs
pBEm1.1443 plasmid was constructed by inserting a 3.7 kb HindIII-EcoRI fragment of plasmid pEm1.1443 (Vicient et al., 2000) into the SmaI restriction site of pSCV1.2. pSCV1.2 was constructed by cloning a 1.7 kb HindIII fragment of plasmid pCaMVNEO (Fromm et al., 1986), containing the nptII gene under control of the CaMV 35S promoter and the NOS terminator, into the plasmid pSCV1. The plasmid pSCV1 was constructed by Dr Glyn A Edwards (Shell Research Ltd) and kindly provided by Dr Tina Barsby (BIOCEM-Nickerson).
Plant material and transformation
pBEm1 constructs were transferred to Agrobacterium tumefaciens LBA4404 by triparental mating. Seeds of Brassica napus cv. Westar were sterilized and plated on germination medium (MS minimal organic medium (Sigma M5519), 30 g l-1 sucrose, 5 g l-1 agargel). Seeds were germinated at 26 °C in 16 h light photoperiod. After 5 d, the cotyledonary petioles (cotyledon with 1 mm petiole) were excised. Single colonies of Agrobacterium tumefaciens strain LBA4404 containing the binary plasmid were grown for 36 h at 28 °C in 2YT medium (Sambrook et al., 1989) supplemented with 5 mg l-1 tetracycline and 50 mg l-1 rifampicine. An aliquot of this suspended culture (0.1 ml) was grown for 14 h at 28 °C in 10 ml of fresh 2YT medium supplemented with the appropriate antibiotics. This bacterial suspension was pelleted by centrifugation for 15 min at 3000 g and resuspended in 10 ml of MS minimal organics medium supplemented with 30 g l-1 sucrose. The cotyledonary petioles were immersed into the bacterial suspension for a few seconds and co-cultivated with Agrobacterium for 48 h on regeneration medium (MS minimal organic medium, 30 g l-1 sucrose, 4 mg l-1 benzyl-amino-purine, 5 g l-1 agargel). After co-cultivation, the cotyledonary petioles were transferred to regeneration medium supplemented with 45 mg l-1 kanamycin and 600 mg l-1 bacteriostatic agent augmentin for 3 weeks. Green shoots were subcultured on shoot elongation medium (MS minimal organic medium, 30 g l-1 sucrose, 5 g l-1 agargel, 45 mg l-1 kanamycin, and 600 mg l-1 bacteriostatic agent augmentin) for 3 weeks. The shoots were transferred to new shoot elongation medium for rooting. As soon as root mass was obtained, the plantlets were transferred in potting mix (40% brown peat, 30% sifted heather, 30% sand) and acclimated to greenhouse conditions in a misting chamber (average relative humidity 84%) for 2 weeks at 21 °C in 16 h light photoperiod. Plants were transferred to a potting mix supplemented with osmocote fertilizer granules (4 g l-1) and grown in the greenhouse at 18 °C. Inflorescences of flowering plants were selfed and isolated with bags. Transgenic seeds were allowed to germinate on germination medium supplemented with 100–150 mg l-1 kanamycin.
Fluorometric measurement and histochemical localization of ß-glucuronidase (GUS) activity
GUS activity was detected using either histochemical staining or fluorometric assay as previously described (Jefferson, 1987). For fluorometric assays, normalization between samples was achieved by protein quantification (Bradford, 1976). Mature embryos dissected from dry seeds were sectioned by hand with a razor blade and X-Gluc stained. Transverse sections of stained cotyledons from 7-d-old seedlings were prepared using cleared and dehydrated tissues embedded in HistoresinTM (Leica Instruments GmbH) according to manufacturer's protocols. 10 micrometre sections were made with a microtome and mounted on a glass slide.
Results and discussion
A construct (pBEm1.1443) containing the -1443 promoter region of AtEm1 gene fused to the uidA reporter gene was prepared and transferred to Brassica napus using Agrobacterium tumefaciens-mediated transformation. Three independently transformed lines were regenerated and their GUS activity examined in T2 plants.
The promoter of the gene AtEm1 of Arabidopsis thaliana is highly active in the embryo during the late stages of seed development (Vicient et al., 2000). In order to determine if the uidA expression in transgenic B. napus was under a similar developmental control, seeds collected from the same stems at different stages of development were assayed for GUS activity (Fig. 1). Samples from plants of the three independently transformed transgenic lines were assayed separately. In the seeds, the GUS activity began to be detectable at the late cotyledonary stage (about 23–33 DAP, days after pollination), and was at a relatively low level until the beginning of the desiccation process (about 45 DAP), with a maximum of GUS activity at the end of desiccation (about 54 DAP). After that, promoter activity decreased slightly during maturity and increased again in dry seeds with an average activity in the three transgenic lines of 260, 305 and 335 pmol 4-MU min-1 mg-1 protein.
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Tests were also carried out for GUS activity at different stages of development in the other tissues of the silique out of the seed (carpels and septum) (Fig. 1
). In these tissues the AtEm1 promoter was active early in the development, with a maximum at mid-cotyledonary stage (about 19 DAP) and decreasing later to an undetectable level at about 30 DAP. GUS assays were also performed in several organs and parts of the plants, including roots, stem, leaves, and flowers at different stages of development. Only flowers showed GUS activity in the fluorometrical assays, with GUS activities of 39, 43 and 50 pmol 4-MU min-1 mg-1 protein at the anthesis stage of development. No detectable promoter activity was found in leaves, stems and roots under these experimental conditions.
In order to have a more detailed localization of the GUS activity some histochemical GUS analysis was performed (Fig. 2). In embryos, the X-Gluc staining was found predominantly in the provascular tissues of the cotyledons and embryo axis, in the central parenchyma of the embryo axis, and in the epidermis of the cotyledons (Fig. 2A, B). AtEm1 promoter was not active in the seed coat or endosperm at any stage of development. In the flowers, the GUS activity was mainly located in the pollen grains (Fig. 2C), but X-Gluc staining was also present in the vascular tissues of petals and stamens (Fig. 2C, D). In floral buds, promoter activity was detected in the vascular tissues of the sepals (Fig. 2E). Pistils did not show GUS activity at any stage of development. Some GUS activity was also detected in the vascular tissues of the caulinar leaves (Fig. 2E). The GUS activity detected in immature siliques was localized in the vascular tissues of the septum and carpels (Fig. 2F, G).
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AtEm1 mRNA disappears very rapidly after germination and the GUS activity directed by this promoter also decreases very quickly in transgenic tobacco and Arabidopsis seedlings after germination (Hull et al., 1996
; Bies et al., 1998; Vicient et al., 2000). In Brassica seedlings, GUS activity also disappears rapidly after germination. The GUS activity in 5 DAG (days after germination) plants represents in average 13% of GUS activity in seeds and this percentage is reduced to 10% in 12 DAG and 5% in 21 DAG. Histochemical assays showed that the distribution of the GUS activity in 7 DAG seedlings was not uniform, and remained for more time in the roots (Fig. 2H) and in the vascular tisssues of the cotyledons (Fig. 2I). The emerging leaves and the apical meristem did not show X-Gluc staining under these experimental conditions. Fluorometrical assays showed that the central parts of the root (vasculature and pericycle) have about 4 times more GUS activity than the external parts (cortex, epidermis and root hairs).
The data presented in this paper demonstrate that the -1443 5' flanking sequence of AtEm1 is capable of directing a high level of GUS activity in the embryos of transgenic Brassica napus. The same fragment was also able to direct GUS activity to the embryos of Arabidopsis and tobacco (Hull et al., 1996; Vicient et al., 2000). The expression of AtEm1 is dependent on the presence of abscisic acid (ABA) and the regulatory protein ABI3 (Parcy et al., 1994). ABA is a phytohormone with similar regulatory properties in all higher plants (Giraudat, 1995). ABI3 is a regulatory protein which has been described in several species (Giraudat et al., 1992; Rohde et al., 1998) with a high level of protein sequence conservation, suggesting that this transcription factor plays a similar role in all of them (Jones et al., 2000; Kurup et al., 2000). ABI3 in Arabidopsis is expressed, among other tissues, in the embryo (Parcy et al., 1994; Rohde et al., 1999, 2000). So, due to the general conservation of ABA responses and ABI3 proteins, is not surprising that the embryo expression of the Arabidopsis AtEm1 promoter is conserved in Brassica and tobacco.
The pattern of GUS activity directed by AtEm1 promoter in embryos of Arabidopsis is not uniform. A higher expression was observed in the provascular tissues of the embryo axis and cotyledons (Vicient et al., 2000). A similar pattern of expression was observed in Brassica napus embryos. However, in tobacco the same promoter directed GUS activity uniformly to the whole embryo (Hull et al., 1996). Taking these observations together, it can be deduced that the factors determining mature embryo expression of the AtEm1 promoter are conserved in all three species, but the factors conferring vascular specific expression are not conserved between tobacco and Arabidopsis. Little is known about the factors determining vascular specific expression in plant genes. Some cis regulatory elements have been determined in the promoter of the French bean (Phaseolus vulgaris L.) grp1.8 gene (Ringli and Keller, 1998) and, although a similar sequence is present in the promoter of the Arabidopsis AtEm1 gene (Vicient et al., 2000), further analysis will be necessary to determine which promoter sequences are involved in the vascular specific expression of AtEm1.
The conservation of the pattern of expression between Arabidopsis and Brassica is not surprising because of the close phylogentic relationships between them, although some differences in the patterns of expression exists: in Brassica, AtEm1 promoter is active in the vascular tissues of some organs in which is not active in Arabidopsis (petals, sepals, green carpels or caulinar leaves). These differences even in so closely related species demonstrate the risks in using heterologous promoters for genetic engineering.
Acknowledgments
This work was supported by the CNRS and by the EC BIOTECH Program (BIO4-CT96-0062) and benefits from the joint CNRS-CSIC Laboratoire Européen Associé Perpignan-Barcelone for Plant Molecular and Cellular Biology. CMV was recipient of a postdoctoral fellowship from the Spanish Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA).
Notes
3 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 
Abbreviations
ABA, abscisic acid; GUS, ß-glucuronidase reporter gene; LEA, late embryogenesis abundant; MS, mineral solution..
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