• In plants, polyamines can generally be synthesized by the ornithine decarboxylase and arginine decarboxylase pathways. However, the model plant Arabidopsis thaliana appears to possess only the arginine decarboxylase pathway. As two paralogous ARGININE DECARBOXYLASE (ADC) genes are present in Arabidopsis, we investigated differential expression and potential differences of promoter activity during seedling development and under specific stress conditions.
• Promoter activities were studied in stable homozygotic transformants harbouring promoter::reporter gene fusions.
• Under temperate conditions, ADC2 promoter activity was strongly associated with seed germination, root and leaf development, whereas ADC1 promoter activity was low during vegetative development. Light, sucrose and ethylene were shown to be important regulators of ADC2 promoter activity. By contrast, in roots and leaves of plantlets subjected to chilling treatment the ADC1 paralogue showed high promoter activity whereas ADC2 promoter activity was considerably decreased.
• In situations of seed germination, root development and response to chilling, the modifications of promoter activities were associated with changes in mRNA levels, emphasizing the involvement of transcriptional regulation in ARGININE DECARBOXYLASE gene expression.
Polyamines, which are present in prokaryotic and eukaryotic organisms (Chang et al., 2000), appear to be important for growth and development of higher plants (Watson et al., 1998; Locke et al., 2000; Hanfrey et al., 2001). In animals and fungi the main synthesis pathway of the diamine putrescine occurs through an ornithine decarboxylase (ODC)-catalysed reaction. However, a membrane-associated decarboxylase has been found to use both arginine and ornithine as substrate in rat brain (Regunathan & Reis, 2000). In plants and some bacteria, distinct ODC-catalysed and arginine decarboxylase (ADC)-catalysed pathways have been thoroughly characterized. After putrescine synthesis, aminopropyl groups are added sequentially to form the triamine spermidine and the tetra-amine spermine.
It is generally considered that, in higher plants, the activities of ODC and ADC undergo distinct regulation depending on developmental and physiological conditions. The ODC activity is generally increased in actively dividing cells (Heimer et al., 1979), and is localized primarily in the meristematic zones (Schwartz et al., 1986). By contrast, ADC is active in elongating cells, in embryonic cells, and in cells under various stress conditions (Flores, 1991). Although nonenzymatic decarboxylation of ornithine can occasionally be detected in Arabidopsis thaliana, neither ODC enzymatic activity nor specific inhibition of the ODC assay has been clearly characterized (Hanfrey et al., 2001). Moreover, genome-wide analysis in Arabidopsis did not identify any intact or degraded ODC sequence, or any ODC expressed sequence tag. Arabidopsis is therefore presently considered to be the only plant, and one of the two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi), lacking ODC activity (Hanfrey et al., 2001).
In this species the formation of putrescine would thus result from ADC-catalysed pathways. Moreover, the corresponding ADC gene appears to have been duplicated at the origin of the Brassicaceae family, thus yielding two paralogues, generally called ADC1 and ADC2 (Galloway et al., 1998). In Arabidopsis, ADC1 and ADC2 are located on chromosomes II and IV, respectively. The protein sequences derived from these two genes show 80% homology. However, the enzyme activities of these proteins may differ somewhat, and analysis of N-terminal sequences indicates that subcellular location could be different (Hanfrey et al., 2001). Characterization of specific roles for ADC and ODC in plants that possess ODC, the absence of ODC in Arabidopsis, and the presence of two distinct ADC in this species emphasize the potential specialization of ADC1 and ADC2 in Arabidopsis.
In order to study these potentially different roles, the promoter activities of ADC1 and ADC2 were studied in stable homozygotic transformants of Arabidopsis harbouring the promoter of either ADC1 (pADC1) or ADC2 (pADC2) fused to the GUS reporter gene. The present work reports the importance of pADC2 activity during seed germination and seedling development, and the importance of pADC1 activity specifically in the response of chilling. Situations of contrasted pADC activity were compared with changes in mRNA levels. Seed germination, root development and response to chilling are thus shown to be characterized by transcriptional activation of ADC genes.
Materials and Methods
Plant material and growth conditions
Arabidopsis seeds of Wassilewskija (WS) ecotype were grown axenically on 1 × Murashige and Skoog medium (M5519, Sigma), 0.8% (w/v) agar, in the absence or presence of sucrose (3%, w/v). Growth was performed under temperate (17/22°C night/day, 16 h light period) or chilling (5/10°C night/day, 14 h light period) conditions. Growth of mature plants was carried out in a growth chamber at 25°C under fluorescent light (16 h light). In the case of salt treatment, plantlets were grown on 1 × MS agar under temperate conditions, then transferred for 4 or 24 h onto 1 × MS agar medium supplemented with 300 mm NaCl. The first generation of stable transformants carrying pADC1::GUS or pADC2::GUS constructs, as previously published (El Amrani et al., 2002), were used to generate the homozygotic transgenic lines used in the present study.
Selection of homozygotic transgenic lines
Transformation of Arabidopsis with pADC1::GUS or pADC2::GUS constructs has been described previously (El Amrani et al., 2002). The ADC1/ADC2 identity of the promoter::GUS fusions was directly confirmed by sequencing, as reported previously (El Amrani et al., 2002). T1 seeds were harvested in bulk and sown on a kanamycin-containing medium (100 µg l−1). Kanamycin-resistant plants were planted on soil, and the T2 seeds were harvested after 2 months from individual T1 plants. The number of loci of integrated T-DNA copies was indicated by segregation of the kanamycin-resistance phenotype in T2 progeny. Transgenic lines showing a kanr : kans ratio of 3 : 1 were considered to be single-locus for the T-DNA insertion. Homozygotic lines of the T3 progeny were used for analysis of promoter activity.
Flowers of eto3 and etr1 ethylene mutants were emasculated with a fine forceps and immediately pollinated. For all the crosses, pADC1::GUS or pADC2::GUS homozygotic lines were used as male parents. Resulting F1 seeds were sown on kanamycin (100 µg l−1)-containing medium. These F1 heterozygous lines expressed both the pADC::GUS transformation and the dominant etr1 and eto3 mutations.
Northern blot analysis
PCR was performed with the primers 5′-AATCGTGGAGAGTTTCGGGT-3′ and 5′-ACCACTCGGATCTGTAACTT-3′ (ADC1) or 5′-CGGTGATGTTTTTATCCCGG-3′ and 5′-TTGCTTGATGAACCATTGGA-3′ (ADC2) which amplify, respectively, a 508 bp fragment from ADC1 (Genbank) U52851 (238–270 bp upstream and downstream of the translation initiation codon, respectively) or a 1171 bp fragment from ADC2 (Genbank) BT000682 (66–1237 bp downstream of the initiation start codon). The resulting fragments were isolated from an agarose gel and cloned into the pGEMT vector (Promega). Distinct digoxigenin-labelled probes were prepared by amplification of DNA fragments in the presence of digoxigenin-11-dUTP (Roche Diagnostics, Germany) as suggested by the manufacturer.
Northern analysis was performed with Dig High Prime DNA labelling and Detection Starter KitII (Roche Diagnostics). Total RNA from Arabidopsis plantlets was extracted with a Total RNA Isolation System kit (Promega). RNA (10 µg) was mixed with sample buffer containing ethidium bromide, separated by electrophoresis through 1% (w/v) agarose gel containing 2% formaldehyde and MOPS 1× and capillary-blotted with 20 × saline sodium citrate (SSC) onto Zeta-Probe GT genomic Tested Blotting membranes (Bio-Rad). RNA was fixed to the membrane by UV.
Prehybridization (2 h) and hybridization (overnight) were performed in hybridization buffer (DIG Easy Hyb, Boehringer Mannheim) at 50°C. After hybridization, membranes were washed for 5 min twice with 2 × SSC, 1% SDS at ambient temperature, and for 15 min twice with 0.1 × SSC, 1% SDS at 50°C. The hybridized probes were immunodetected with an alkaline phosphatase-conjugated antidigoxigenin antibody and visualized with chemiluminescence substrate (CSPD, Roche) following the supplier's instructions (Roche Diagnostics).
Histochemical GUS staining was performed as described previously by Jefferson et al. (1987). Plant tissues containing pADC1::GUS or pADC2::GUS fusions were stained for GUS activity at 37°C overnight, unless otherwise indicated in figure legends. Tissues were washed with sodium phosphate buffer (50 mm, pH 7) then left overnight in 70% ethanol. No background staining was observed in any kanamycin-sensitive plants.
Characterization of homozygotic promoter::GUS transgenic lines
Stable transformants of Arabidopsis heterozygotic lines carrying pADC1::GUS or pADC2::GUS constructs (El Amrani et al., 2002) were used to generate homozygotic transgenic plants. In the present work, only homozygotic lines from the T3 progeny were used for analysis of reporter gene expression as described in Materials and Methods. At least 10 independent transgenic lines were analysed for each construct. Despite the expected variability caused by the position effect, all pADC2::GUS transgenic homozygotic lines displayed similar patterns of reporter GUS expression. By contrast, pADC1::GUS transformation resulted in a significant (> 40%) proportion of homozygotic kanamycin-resistant lines showing no GUS staining in a wide range of growth conditions. The pADC1::GUS lines showing maximum GUS staining were chosen for subsequent experiments.
The pADC1::GUS and pADC2::GUS homozygotic transgenic lines showed contrasting patterns of reporter gene expression in flowering plants which had grown under temperate conditions. In pADC1::GUS lines, high levels of GUS staining were found in stigmata and staminae and particularly in pollen grains, whereas pADC2::GUS lines showed no significant promoter activity in pollen grains. These pADC2::GUS lines showed more generalized expression in floral organs and strong GUS staining in the stigmata. Thus reporter gene expression in floral organs from homozygotic lines (results not shown) was similar to previous results on heterozygotic lines (El Amrani et al., 2002).
Previous analysis of the 1500 bp proximal regions of ADC1 and ADC2 promoters had shown that they had a low level of homology and possessed a complex array of putative cis-acting regulatory elements (El Amrani et al., 2002). Moreover, one specific feature of pADC1 was shown to be the presence of a copy of a transposable element (El Amrani et al., 2002). Further analysis was carried out using the PlantCARE database (Lescot et al., 2002) to identify putative plant regulatory elements, especially those showing a contrasting pattern of presence/absence between pADC1 and pADC2. The transcription initiation sites of ADC1 (Accession No. U52851) and ADC2 (Accession No. BT000682) were deduced from the latest issues of cDNA sequences (October 2002, National Center for Biotechnological Information), and are indicated in the nucleotide sequences of the promoter regions (Fig. 1) by position +1. The ATG start codons of ADC1 and ADC2 are, respectively, 382 and 519 bp downstream from the transcription initiation sites. The pollen-specific regulatory element AAATGA (Weterings et al., 1995) was present six times in both orientations in pADC1, and only once in pADC2 (Fig. 1). Multiple copies of cis-acting regulatory elements (Argüello-Astorga & Herrera-Estrella, 1996) involved in light responsiveness were present in both promoters (data not shown). Whereas pADC2 presented five sucrose-responsive elements (SURE), pADC1 contained only one SURE element (Fig. 1). The ADC1 promoter, but not the ADC2 promoter, contained two copies of cis-acting regulatory elements (LTR) involved in the response to low temperature (Fig. 1). The dehydration-responsive element (DRE) was found only in the ADC1 promoter, and STRE, a stress-responsive element, was found in both promoters (Fig. 1). Both ADC1 and ADC2 promoters contained ERE, an ethylene-responsive element (Fig. 1). Thus a number of relevant cis-acting regulatory elements such as SURE, LTR and pollen-specific regulatory elements showed a contrasting pattern of distribution between pADC1 and pADC2.
Activities of pADC1 and pADC2 during seed germination and early vegetative growth
Whereas pADC2, but not pADC1, activity was high in mature siliques, mature and dry seeds showed no activity of pADC1 and pADC2 (Fig. 2a). However, during sensu stricto seed germination from imbibition up to emergence of the radicle (Bewley, 1997), GUS histochemical analysis showed a contrasting pattern of activity between pADC1 and pADC2. No GUS staining was observed in pADC1::GUS transgenic lines (Fig. 2b). By contrast, pADC2::GUS transgenic lines showed strong activity which started at an early stage of imbibition with staining of testa, cotyledons and embryonic axes (Fig. 2b). High activity of pADC2 was then detected in the emerging radicle (Fig. 2b). The transcriptional activation of pADC2 promoter was therefore characteristic of the germination process.
Analysis of reporter GUS expression during seedling growth and leaf emergence was carried out under conditions of optimal growth under temperate conditions. To obtain optimal root development (Jones & Grierson, 2003), plantlets were grown in the presence of 3% sucrose (Fig. 3). Whereas in pADC1::GUS transgenic lines light GUS staining was detected in cotyledons and limbs of first leaves (Fig. 3a), pADC2::GUS transgenic lines showed strong GUS staining in cotyledons and in both petioles and limbs of first leaves (Fig. 3d).
No pADC1-driven GUS staining was detectable in any part of the root system, in either the primary root or lateral roots (Fig. 3b). By contrast, the ADC2 promoter was highly active in roots (Fig. 3d), especially in the root apical meristem, with decreasing activity in subapical and differentiating regions of the root, except in vascular tissues where GUS staining was maintained at high level. Closer histochemical analysis during root development showed that pADC2 promoter activity was closely related not only to the apical meristem, but also to lateral root primordium (LRP) formation (Fig. 3e–j). Activity of pADC2 was high in the pericycle cells of xylem poles during the first anticlinal divisions that initiate LRP formation (Fig. 3e). By contrast, pericycle cells at the opposite side of the stele showed significantly lower pADC2 activity (Fig. 3e). The pADC2 activity remained high after the start of periclinal divisions in the LRP, and during the eight different stages of lateral root development described by Malamy & Benfey (1997), until emergence from the parent root (Fig. 3e–j). After emergence, reporter gene expression in pADC2::GUS transgenic lines was maintained in the axis of the secondary root, especially in the apical zone, as occurred in the primary root.
Effects of light and sucrose on the activities of pADC1 and pADC2
Dark growth of seedlings in the absence of sucrose under temperate conditions resulted in the absence of GUS staining in roots and shoots of pADC1::GUS (Fig. 4e,f) and pADC2::GUS (Fig. 4a,b) transgenic seedlings. These results strongly contrasted with pADC2 activity in light-grown seedlings in the absence (Fig. 4i) and presence (Fig. 3c) of sucrose. However, sucrose treatment of dark-grown seedlings restored GUS staining in both roots and shoots of pADC2::GUS transgenic lines (Fig. 4c,d), showing that pADC2 was highly responsive to sucrose and indicating that sucrose may also be a component of ADC2 induction by light. By contrast, sucrose treatment did not modify GUS staining in roots of dark-grown pADC1::GUS transgenic seedlings, but gave a slight activation of pADC1 in cotyledons (Fig. 4g,h).
Effects of chilling on activities of pADC1 and pADC2
When germination and seedling growth were carried out at low temperature (5°C night, 10°C day) the relative pattern of pADC1 and pADC2 activities was greatly affected, with pADC2 activity significantly decreasing in roots and leaves (Fig. 4i–l), whereas pADC1 activity increased in leaves and was induced in roots (Fig. 4m–p). The latter effect was particularly striking. When chilling treatment was applied to plantlets that had previously grown under temperate conditions, strong GUS staining was observed in roots of pADC1::GUS transgenic lines after 24 h treatment (data not shown), whereas under temperate conditions these lines presented no detectable GUS staining in roots. By contrast, saline stress (300 mm NaCl) for 4 h under conditions that have been shown to induce gene responses (Knight et al., 1997) and ADC expression (Gong et al., 2001) in the Col0 genetic background did not modify ADC promoter activities, either in pADC1::GUS (Fig. 3s,t) or in pADC2::GUS transgenic lines (Fig. 4q,r). Longer salt treatment (24 h) did not result in any increase of promoter activity (data not shown).
Activities of pADC1 and pADC2 in ethylene-response mutant backgrounds
Given the importance of ethylene in germination and seedling growth (Leon & Sheen, 2003), and the link between ethylene and polyamine synthetic pathways (Locke et al., 2000), transcriptional activity of ADC promoters was investigated in ethylene-response mutant backgrounds (Fig. 5a–e) under conditions of maximal pADC2 activity (Figs 3, 4). The transgenic pADC::GUS lines were crossed with etr1, an ethylene-insensitive mutant impaired in ethylene perception and signal input (Schaller & Bleecker, 1995), or with eto3, a mutant that overproduces ethylene in etiolated seedlings (Woeste et al., 1999). The latter mutant shows a characteristic ethylene-induced ‘triple-response’ phenotype in a constitutive manner (Guzman & Ecker, 1990). The progeny were heterozygous for the T-DNA and for eto3 and etr1 dominant mutations, which allowed us to use the F1 generation for ADC transcriptional expression. The transgenic pADC::GUS ×eto3 lines exhibited the triple response when growth was carried out in the dark. Low or no GUS staining was observed during dark growth in the presence of sucrose in the pADC1::GUS lines, and in the progeny obtained from crosses between pADC1::GUS and both etr1 and eto3 mutants (data not shown). By contrast, pADC2::GUS × etr1 lines showed significantly decreased GUS activity under illuminated (Fig. 5a,b) and dark (Fig. 5c,d) conditions, indicating that perception and transduction of the ethylene signal may be involved in transcriptional regulation of pADC2. No significant variation of GUS expression was observed when pADC2::GUS fusion was expressed in the eto3 mutant background (Fig. 5c,e).
Differential accumulation of ADC1 and ADC2 mRNA
ADC1 and ADC2 mRNA levels were investigated by Northern blot analysis in situations of contrasted promoter activities in the absence of sucrose. High ADC2 promoter activity and undetectable ADC1 promoter activity during germination (Fig. 2b) were associated with high levels of ADC2 mRNA and very low levels of ADC1 mRNA in imbibed seeds, as shown in Fig. 6(a). Both ADC1 and ADC2 mRNA were found in 15-d-old plantlets grown under temperate conditions (Fig. 6b). This was consistent with the detection of both ADC1 and ADC2 promoter activities in leaves under temperate conditions (Fig. 4i,m). However, the contrasting activities of ADC1 and ADC2 promoters (Fig. 4i,m) did not result in highly contrasting levels of mRNA (Fig. 6b), although repeated Northern analysis showed consistently higher levels of ADC2 mRNA than of ADC1 mRNA (data not shown). Northern blot analysis was also carried out in plantlets grown at low temperature. By contrast with temperate conditions, enhanced ADC1 promoter activity at low temperature (Fig. 4o,p) was associated with a strong increase of ADC1 mRNA levels. Conversely, ADC2 mRNA levels decreased at low temperature (Fig. 6c) in accordance with lower pADC2 activity (Fig. 4k,l).
Differential activity of the promoters of ADC1 and ADC2
The existence of two ADC genes in Arabidopsis (Galloway et al., 1998) is in agreement with the duplicated status of its genome (Arabidopsis Genome Initiative, 2000). The stabilization of genome organization has provided the opportunity for functional divergence of the two paralogues. Although the protein sequences of ADC1 and ADC2 show a high degree of homology, the possibility of divergence in terms of substrate specificity and enzymatic regulation cannot be discounted. Biochemical analysis of mammalian ADC has shown that it could use both ornithine and arginine, although the specific ODC inhibitor, difluoromethylornithine, had no effect on enzyme activity with either substrate (Regunathan & Reis, 2000). Previous studies have emphasized the hypothesis that Arabidopsis ADC1 and ADC2 may be targeted to different subcellular localizations (Hanfrey et al., 2001). This implies action on distinct metabolic pools and thus strongly suggests important functional differences. In addition to possible variation of protein and enzyme functions between ADC1 and ADC2, important developmental and stress-response modifications of mRNA expression between ADC1 and ADC2 in Arabidopsis have been reported recently (Soyka & Heyer, 1999; Perez-Amador et al., 2002; Piotrowski et al., 2003).
Many studies have insisted on the importance of post-transcriptional and post-translational regulation of ADC accumulation and activity (Malmberg & Cellino, 1994; Borrell et al., 1996; Watson & Malmberg, 1996). Thus in Arabidopsis plantlets stressed by potassium deficiency, ADC mRNA did not correlate with increase of ADC enzyme activity (Watson & Malmberg, 1996). However, in other situations, such as response to acid and salt stresses, ADC mRNA levels were found to correlate with ADC activity (Perez-Amador et al., 1995; Chattopadhyay et al., 1997). However, analysis of ADC1 and ADC2 promoters showed striking differences, such as various cis-acting regulatory elements, and the presence of a transposable element specifically in the promoter of ADC1 (El Amrani et al., 2002). In the present study analysis of homozygotic pADC::GUS transgenic lines confirmed that in imbibed seeds, seedlings, roots, stems, leaves and flowers under temperate conditions, pADC1 activity was significantly more development- and tissue-limited than pADC2, which had a more general pattern of activity. The more restricted pattern of activity for pADC1 may be related to the presence of the transposable element through disruption of cis-acting regulatory elements, as has recently been shown for the Zea mays a1 gene (Pooma et al., 2002), or through epigenetic regulation. Introduction of the pADC1::GUS transgene resulted in over 40% of silenced lines, whereas pADC2::GUS lines all showed reporter gene activity. The ADC1 promoter is likely to be more sensitive to epigenetic regulation as its sequence contains a transposable element that is present in nearly 2000 copies in the Arabidopsis genome (El Amrani et al., 2002). In plants and animals DNA methylation is involved in heritability and flexibility of epigenetic states, and it has been shown that transposable elements are the primary targets of genomic DNA methylation (Okamoto & Hirochika, 2001). As insertion of transposable elements may influence expression of neighbouring genes via DNA methylation, epigenetic regulation may be one of the mechanisms controlling ADC1 expression.
However, situations of highly contrasting promoter activity were shown to correlate with corresponding variation of mRNA levels. Thus Piotrowski et al. (2003) have reported ADC2 mRNA to be at much higher levels than ADC1 mRNA in roots. This is, at least partially, consistent with the differential activities of ADC1 and ADC2 promoters in roots of plants grown under temperate conditions (Fig. 3b,d). By contrast, in young leaves and rosette leaves both pADC1 and pADC2 showed significant activity (Fig. 3a,d), and both ADC1 and ADC2 mRNA have been shown to be present (Perez-Amador et al., 2002; Piotrowski et al., 2003). Detection of pADC1 and pADC2 activities in inflorescences was also associated with the detection of ADC1 and ADC2 transcripts in flowers and siliques (Soyka & Heyer, 1999). Thus these various relationships between ADC1 and ADC2 promoter activities and mRNA levels strongly suggested that both promoter sequences were functional. Moreover, the reporter gene strategy had previously shown that pADC1, but not pADC2, was highly active in pollen grains (El Amrani et al., 2002). This was partially correlated with the presence of the sequence AAATGA, which has been described as capable of driving pollen-specific expression independently of orientation (Weterings et al., 1995). This sequence was present six times in both orientations in the ADC1 promoter, and only once in the ADC2 promoter. This provided strong evidence that evolution had selected divergent cis-acting regulatory elements in the promoter sequences of the two ADC paralogues, and that these divergent elements were functional. Moreover, this strongly indicated that the presence of the transposable element did not interfere negatively with this pollen-specific expression, and may even contribute to pollen-specific expression through the presence of an additional AAATGA sequence (Fig. 1). Finally, the striking differences of tissue-specific promoter activity clearly showed that ADC genes were not transcribed at a basic level in all tissues, with subsequent regulation at post-transcriptional and post-translational levels, and correlatively suggested that transcriptional control may be important for ADC gene expression.
High ADC2 gene expression during seed germination and seedling growth under temperate conditions
Seed maturation is followed by embryo developmental arrest during seed dehydration; on germination, embryo arrest is lifted and cell division resumes (Raz et al., 2001). These developmental stages are highly regulated and are of great importance in the life cycle of monocarpic species such as Arabidopsis. The ADC paralogues were found to show contrasting promoter activities during these developmental stages. None of these genes showed activity during seed maturation and dehydration. During germination, which starts with uptake of water by the quiescent dry seed and terminates with emergence of the embryonic axis (Bewley, 1997), no ADC1 promoter activity was observed, whereas ADC2 promoter activity was high as early as 10 h after seed imbibition, and before primary root emergence (Fig. 2b). This correlated with the detection of significant levels of ADC2 mRNA in imbibed seeds (Fig. 6a). It has been shown that germination of barley seed was promoted by addition of exogenous polyamines (putrescine, spermidine and spermine), and it is suggested that endogenous polyamines may play a growth-promoting role complementary to ethylene in the normal course of barley germination (Locke et al., 2000). At the end of the germination process, ADC2 promoter activity was high in the emerging radicle. Involvement of ADC in germination and radicle extrusion is in agreement with the general idea that ADC is active in elongating cells (Nam et al., 1997). The specific reason why the ADC2 gene is upregulated during this process remains to be elucidated.
Under temperate conditions, pADC2 was highly active in seedling and plantlet roots, which is in accordance with the higher level of ADC2 transcripts in roots (Piotrowski et al., 2003). A high level of ADC transcripts in roots is also in general agreement with high ADC activity in roots (Hanfrey et al., 2001) and a high level of putrescine in roots (Watson et al., 1998; Piotrowski et al., 2003). Moreover, the pattern of pADC2::GUS activity reveals a tight relationship with rhizogenesis processes. Lateral root formation involves the stimulation of pericycle cells to proliferate and create a new root meristem. Formation of the lateral root primordium may be divided into different stages that can be characterized by histology, cell division patterns, and gene expression (Malamy & Benfey, 1997). Activation of pADC2 was found to be induced at stage I of primordium development, before the first periclinal division occurs. At later stages of development, strong GUS staining was observed in the whole primordium. Arabidopsis spe mutants, which are characterized by low levels of ADC activity, are deeply affected in root development, with enhanced lateral root formation in single mutants and a compact root system in double mutants (Watson et al., 1998). A number of previous studies have associated ADC activity with root growth and root branching (Biondi et al., 1993; Watson et al., 1998; Hummel et al., 2002). The present study shows that this association is correlated with activation of the ADC2 promoter in the early stages of lateral root formation. However, to our knowledge no study has yet addressed the mechanisms of polyamine action on root development.
Activation of both ADC1 and ADC2 promoters was found in the aerial parts of plantlets, with pADC2 showing much higher activity than pADC1. ADC1 and ADC2 mRNA levels have been reported to be equivalent in plantlet leaves (Piotrowski et al., 2003). Similarly, ADC1 and ADC2 mRNA levels in whole plantlets showed much less contrast (Fig. 6b) than the differences in promoter activity (Fig. 4i–n). Thus important post-transcriptional regulation probably acts on ADC1 and ADC2 transcripts. Expression of ADC2 gene in stems is in accordance with increased ADC mRNA levels and increased ADC activity in hypocotyls of germinating soybean (Nam et al., 1997). Expression of ADC1 and ADC2 genes in leaves is in accordance with the physiological importance of polyamines in leaves for photosynthetic activity (Chang et al., 2000). At least one of the ADC proteins is likely to be targeted to the chloroplast (Perez-Amador et al., 2002), and the oat ADC has been shown to be localized in the chloroplast (Borrell et al., 1995). Moreover, light was found to be a strong inducer for ADC1 and ADC2 promoter activity, with no or little promoter activity during dark growth (Fig. 4a–n). This strong induction by light was consistent with the presence of many putative light-responsive cis-acting regulatory elements in the promoters of both ADC1 and ADC2. Similarly, the promoter region of the carnation ADC gene is rich in light-responsive, cis-acting regulatory elements, and ADC transcripts increase tenfold after light exposure (Chang et al., 2000). In the case of ADC2, promoter activity was also increased in the dark in the presence of exogenous sucrose, especially in roots (Fig. 4a–d). This was consistent with the presence of numerous sucrose-responsive, cis-acting regulatory elements in the promoter of ADC2. It is thus tempting to speculate that this induction by sucrose, resulting in high pADC2 activity in roots, may be part of shoot–root relationships during light growth of plants through the transport of sucrose from shoot to root.
The pattern of pADC2 activity during seedling development was found to be affected in dominant ethylene-mutant backgrounds, even in the presence of light and sucrose, which normally resulted in high pADC2 activity (Fig. 3). Gallardo et al. (2002) have recently highlighted the importance of S-adenosylmethionine synthesis for seedling development of Arabidopsis. This is consistent with an essential role of endogenous ethylene during seedling development, which has also been described in other species (Petruzzelli et al., 2000). Thus ADC2 would be part of an array of genes activated by ethylene during seedling development.
High ADC1 gene expression in response to chilling
Chilling, which has been shown to increase ADC activity and polyamine levels (Lee 1997; Shen et al., 2000; He et al., 2002) had a strong effect on ADC1 and ADC2 promoter activity. Strikingly, growth under chilling conditions thoroughly modified the respective patterns of pADC1 and pADC2 activity, with pADC1 and pADC2 becoming, respectively, highly active and poorly active in roots (Fig. 4i–p). This strong effect appeared to be a specific response to chilling rather than a general response to stress, as salt treatment did not change the patterns of ADC1 and ADC2 promoter activity. The chilling effect was correlated to changes in mRNA level, and consistent with the specific presence of two copies of a low-temperature response element in the promoter of ADC1 and with the potential impact of the transposable element on gene expression, as a copy of this low-temperature-response element is part of the ADC1 transposable element (Fig. 1). Moreover, changes in temperature may also affect epigenetic control of ADC1 promoter activity. Further work should therefore determine whether this ADC1/ADC2 functional divergence derives directly from the presence of the transposable element in the ADC1 promoter.
The functional role of ADC activity in response to chilling is highlighted by previously reported correlations between agmatine accumulation and frost resistance in wheat (Racz et al., 1996), and by the involvement of polyamines and increased ADC activity in chilling tolerance of cucumber (Shen et al., 2000). In Arabidopsis, D-Arg inhibition of ADC drastically reduced plantlet development at low temperature and caused symptoms of chilling injury (data not shown). Thus in Arabidopsis the polyamine response to chilling is shown to correlate with transcriptional activation of the ADC1 promoter. This switching effect of chilling on ADC1 and ADC2 relative expression therefore provides a good experimental model for understanding the specific roles of ADC1 and ADC2.
We thank the Nottingham Arabidopsis stock center for providing the eto3 and etr1 ethylene mutants.