JXB Advance Access originally published online on June 30, 2006
Journal of Experimental Botany 2006 57(10):2455-2469; doi:10.1093/jxb/erl019
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Expression profile of CBF-like transcriptional factor genes from Eucalyptus in response to cold
Université Paul Sabatier: UMR 5546, ‘Surfaces Cellulaires et Signalisation chez les Végétaux’ Pôle de Biotechnologie Végétale, 24 Chemin de Borde Rouge, BP 42617 Auzeville, F-31326 Castanet-Tolosan, France
To whom correspondence should be addressed. E-mail: teulieres{at}scsv.ups-tlse.fr
Received 4 November 2005; Accepted 10 April 2006
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Abstract |
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Two CBF (CRT/DRE-binding factor) homologues isolated from Eucalyptus gunnii were designated EguCBF1a and EguCBF1b and belong to a gene family which includes at least five members. Both promoter and coding sequences were found to exhibit the main characteristics of a CBF transcription activator gene and, as expected, the corresponding protein targeted the nucleus. Gene expression was quantitatively analysed using real-time reverse transcription–polymerase chain reaction (RT–PCR) after a short exposure to different environmental conditions or along a two-step cold acclimation programme with either short or long daylengths. A very strong and fast response to cold was observed, with dark conditions and cold intensity (down to 0 °C) having a positive effect on the magnitude of induction. The two genes under study exhibited several similar features such as light response. However, interestingly, their regulation by cold proved differential and complementary as EguCBF1a was more transiently induced by a direct and intense exposure while EguCBF1b responded to milder treatments and exhibited a longer (i.e. which started earlier and finished later) time course. During acclimation, the short daylength positively affected the freezing tolerance in the same way as it positively affected the CBF transcript accumulation, suggesting a potential involvement of these genes in the adaptive response. Although very quick after the first signal, the up-regulation of the two EguCBF1 genes unexpectedly lasted throughout the chilling culture, and new inductions were seen during the thermoperiod transitions. Using a quantitative and highly sensitive measurement of gene expression combined with the application of a cold treatment consistent with natural environmental conditions, this study provides new information on the regulation of CBF-like genes by cold in planta.
Key words: CBF/DREB1 transcription factor, cold acclimation, Eucalyptus, freezing tolerance, gene expression, real-time RT–PCR
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Introduction |
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Cold, drought, and high salinity, which all cause dehydration damage to the plant cell, are the most common environmental stresses that influence plant growth and development, limiting productivity in cultivated areas worldwide. In the last decade, numerous investigations have focused on the identification of stress-regulated genes, with the aim of better understanding their functions in stress tolerance mechanisms (Thomashow, 2001; Zhu, 2002; Seki et al., 2003; Shinozaki et al., 2003) and to provide the basis for efficient strategies in plant breeding programmes.
Due to its fast growth and fibre quality, Eucalyptus is the most commonly planted hardwood in the world, in particular for paper making. However, although widely distributed, its extension is mostly restricted to southern areas because of freezing sensitivity. In the absence of any physiological or morphological adaptive strategy to avoid frost, overwintering of this perennial species essentially depends on the capacity of cells to tolerate apoplastic freezing. Eucalyptus, as well as many other plant species, develops increased freezing tolerance in response to low but non-freezing temperatures. This adaptive response, known as cold acclimation, takes place on the time scale of days or weeks as a result of a combination of physiological and metabolic changes depending on transcriptome modifications (Thomashow, 1999).
At present, the best understood genetic system with a role in cold acclimation is the Arabidopsis CBF (CRT/DRE-binding factor) cold response pathway. The CBF/DREB1 proteins belong to the AP2/ERF DNA-binding protein family (Riechmann and Meyerowitz, 1998), which includes >140 members in Arabidopsis thaliana. CBF gene expression is induced within 1 h by low temperature, and the encoded transcriptional factors in turn activate the expression of many cold-responsive genes (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997). The CBF regulon comprising several dozen genes (Seki et al., 2001; Fowler and Thomashow, 2002; Cook et al., 2004; Gilmour et al., 2004; Maruyama et al., 2004) represents up to 12% of the cold-regulated genes (Fowler et al., 2002; Cook et al., 2004). Moreover, when constitutively overexpressed in Arabidopsis, CBF genes induce the expression of the downstream genes (CBF regulon) under non-stress conditions and confer freezing, drought, and salt tolerance to the corresponding transgenic plants (Gilmour et al., 1998; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999). In addition, the CBF cold response pathway was found to play a prominent role in the configuration of the low temperature metabolome of Arabidopsis (Cook et al., 2004). Therefore, this class of genes represents a critical component in signal transduction of cold acclimation.
Highly conserved in plants, CBF orthologues exhibiting the same characteristic elements as Arabidopsis thaliana have been found in several plant species, including herbaceous species such as Brassica napus (Jaglo et al., 2001), Lycopersicon esculentum (Hsieh et al., 2002), Oryza sativa (Dubouzet et al., 2003), and Capsicum annuum (Kim et al., 2004; Hong and Kim, 2005), as well as the woody plant Prunus avium (Kitashiba et al., 2004). Moreover, as in Arabidopsis, overexpression of two Brassica CBF-like genes in Brassica napus allowed freezing tolerance to increase (Savitch et al., 2005). However, despite this apparent high conservation, some structural and regulatory differences were observed in the CBF cold response pathway among the plant species. For example, the CBF regulon of L. esculentum (a chilling-sensitive species) was found to be smaller and less diverse in function than the regulon of frost-tolerant plants such as A. thaliana, which partly explains the differences in plant behaviour in low temperatures (Zhang et al., 2004). Another recent work on wheat showed that several CBF genes have dramatically different levels of induction after cold exposure linked to the frost tolerance of the corresponding recombinant lines (Vagujfalvi et al., 2005). It could therefore be hypothesized that the CBF pathway is more or less complete, depending on the complexity of the cold-tolerance mechanisms for the considered plant species.
Although recent breakthroughs have increased the knowledge of the molecular basis of frost hardiness and the CBF pathway in herbaceous species, very little is known concerning woody plants. To date, the only published information on a CBF gene from woody plants is a functional analysis of PaCBF (Kitashiba et al., 2004) from wild cherry by heterologous expression in Arabidopsis. The present study reports the molecular characterization and the transcriptional patterns of two CBF-like genes, EguCBF1a and EguCBF1b, isolated from Eucalyptus gunnii. The real-time reverse transcription–polymerase chain reaction (RT–PCR) method was used to quantify sensitively the influence of various conditions of cold culture (light, photoperiod, intensity, or duration of cold treatment) on the expression of both EguCBF1a and b. The experimental system was completed by the design of two chilling programmes imitating natural conditions and leading to a differential level of freezing tolerance measured by ion leakage. Among the numerous data, the detailed kinetics of CBF transcript abundance during a cold culture programme, aligned with the level of freezing tolerance, suggest that the two EguCBF1 genes are involved in the development of Eucalyptus cold acclimation.
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Materials and methods |
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Plant material
Two Eucalyptus lines from a plant breeding programme managed by AFOCEL were studied. The E. gunnii cell suspension culture from line 634, maintained as previously described (Teulières et al., 1989), was used for gene isolation, Southern analysis, and protoplast isolation. Plantlets from a hybrid Eucalyptus gunniixEucalyptus dalrympleana (‘E. gundal 208’) were provided by TEMBEC R&D KRAFT as material for gene expression studies. The 1-year-old plantlets were grown in controlled-environment chambers at 25 °C day/22 °C night, with a long-day (LD) photoperiod (16 h/light=115 µmol m–2 s–1 supplied by Lumilux Daylight 58 W Osram).
Gene isolation and sequence analysis of EguCBF1
Based on the sequence similarity between the published plant CBF/DREB genes, two degenerate primers (Table 1) were designated from the conserved regions to amplify the CBF orthologues from E. gunnii, using the High-Fidelity PCR system (Roche). The resulting product, cloned in the pGEM-T Easy vector (Promega, France), was identified as CBF-like using BLAST analysis and designated EguCBF1. The isolation of the promoter sequence and complete ORF (open reading frame) was carried out using a Universal Genome Walker Kit (Clontech, USA) on 10 µg of E. gunnii genomic DNA digested by four blunt-end-generating restriction enzymes (EcoRV, DraI, PvuII, and StuI). After purification, the restriction fragments were ligated with Genome Walker adaptors. The first PCR was performed on each restriction fragment set using an EguCBF1 sequence-specific primer (EguCBF1a-ext.5', Table 1) and the adaptor primer 1 (5'-GTAATACGACTCACTACTAGGGC-3'). Using another EguCBF sequence-specific primer (EguCBF1a-int-5', Table 1) and the adaptor primer 2 (5'-ACTATAGGGCACGCGTGGT-3'), the second PCR amplification was performed on the products of the first PCR. Finally, the PCR products were cloned into pGEM-T Easy vector and mobilized into Escherichia coli (DH5
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In the second round, the EguCBF1 sequence was used for BLAST analysis against the Eucalyptus cold cDNA library available in the laboratory (unpublished data). The result allowed the identification of a matching clone which was presumed to be a part of another CBF1-like gene. As previously described for the first EguCBF1 expressed sequence tag (EST), the extension of the upstream sequence of this EST was carried out. The comparison between the two sequences led to the conclusion that two different CBF1-like genes had been isolated, which were designated as EguCBF1a and b, respectively.
Promoter sequence analysis was performed on the EguCBF1a sequence using the PLACE Signal Scan Search program (Prestridge, 1991; Higo et al., 1999), (http://www.dna.affrc.go.jp/PLACE/signalscan.html). The alignment of the CBF/DREB1 proteins was performed using ClustalW, and shading was done using Esprit 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The phylogenetic tree was constructed using TreeTop-Phylogenetic Tree Prediction, (http://www.genebee.msu.su/services/phtree_reduced.html).
Southern blotting analysis
A 10 µg aliquot of genomic DNA was isolated from E. gunnii using the DNeasy Plant Maxi kit (Qiagen) and digested with BamHI, EcoRI, EcoRV, or HindIII enzymes. The restriction fragments were separated by electrophoresis in a 0.8% agarose gel, then blotted onto a nylon membrane filter (Amersham). Under high-stringency conditions, the membrane was successively hybridized to two different 32P-labelled probes: the first one was the CBF full-length ORF sequence and the second was a partial EguCBF1 sequence corresponding to the 3'-terminal region (DSAWR to stop codon). The second hybridization on the stripped filter was performed in order to improve hybridization specificity and avoid putative cross-hybridization between the CBF coding sequence probe and the numerous genomic sequences which contain the highly conserved ERF/AP2 DNA-binding domain present in the ORF.
Protoplast isolation and transient expression of EguCBF1::GFP fusion proteins
The coding sequences of EguCBF1s were cloned in-frame with the green fluorescent protein (GFP) into the pGreen vector (Hellens et al., 2000) as a C-terminal fusion expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The ORFs were amplified by PCR (Expand High Fidelity, Roche) using the following primers: EguCBF1a-XmaI (5'-TAACCCGGGATGAACCCTTTCTCTTCTCATTCCCAT-3') and EguCBF1a-NcoI (5'-TAACCATGGAGATGGAATAGCTCCATAATGACGTGTG-3') or EguCBF1b-BamHI (5'-AATAGGATCCATGAACTCTTCCTCTTATATCTCCCAT-3') and EguCBF1b-NcoI (5'-AATTCCATGGTTGGAATAGCTCCATAATGACGTGTACGC-3'). The corresponding ORFs of EguCBF1a and b were cloned using the XmaI–NcoI and BamHI–NcoI restriction sites of the pGreen vector, respectively.
Protoplasts were obtained from E. gunnii cell suspension culture and transfected using a polyethylene glycol method as previously described (Teulières et al., 1989). A 0.3 ml aliquot of protoplast suspension (0.5x106) was transfected using 50 µg of sheared salmon sperm DNA as carrier and 30 µg of EguCBF1a::GFP or EguCBF1b::GFP or GFP (control) plasmid DNA. After transfection, the protoplasts were incubated for 16 h at 25 °C and then analysed for GFP fluorescence (500–520 nm) using confocal microscopy. All transient expression assays were performed in three independent experiments.
Cold treatments
All the results were obtained from at least two independent experiments on two or three plantlets of E. gundal, depending on the treatment. For the corresponding gene expression studies, total RNA was extracted from a pool of leaves randomly harvested on the plantlets.
First, the time-course of EguCBF1 transcript production was studied at 15 min, 30 min, 2 h, 5 h, and 24 h, on two plantlets grown at 4 °C in the dark or in continuous light (45 µmol m–2 s–1 supplied by Lumilux Daylight 58 W Osram). Then, the effect of the cold shock intensity on the EguCBF1 transcription rate was evaluated on the plantlets after transferring them directly from 22 °C/night to lower temperatures (12, 8, 4, 0, or –4 °C) for 2 h in the dark.
For cold acclimation, the chilling programme consisted of two steps: 4 d at 12 °C/day and 8 °C/night followed by 6 d at 4 °C day and night (Fig. 1). In two different experiments, this chilling programme was coupled with a photoperiod corresponding either to a long daylength (LD=16 h light) or a short daylength (SD=8 h light). Before the acclimation programme, the plantlets were cultured with the appropriate photoperiod for 3 d, and the thermoperiod transition was started at the same time as the light was switched on (from 22 °C/night to 12 °C/day and then 8 °C/night to 4 °C/day). In addition, in order to avoid photoinhibition stress on plants, the light intensity was reduced in the culture chamber during cold treatment (45 µmol m–2 s–1 supplied by Lumilux Daylight 58 W Osram). Frost tolerance was evaluated on day 4, day 7, and day 10 by measuring the electrolyte leakage from leaf discs after freezing at –2 °C h–1, using previously described methods (Leborgne et al., 1995). The measurement was expressed as the percentage of cell viability after freezing at –6 and –8 °C compared with the viability of unfrozen leaf discs. It was given as the mean of at least three plantlet measurements.
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For expression studies during the acclimation programme, RNA was extracted from a pool of leaves randomly harvested on the three plantlets at the indicated times (Fig. 1): (i) 30 min, 2 h, and 5 h after the temperature changes from 22 °C to 12 °C (day 1) or from 8 °C to 4 °C (day 5); (ii) 15 min before and 2 h after the daily thermoperiod transition (12 °C day/8 °C night) for SD culture conditions; and (iii) 24 h for each day except days 8 and 9.
Abiotic stress treatments and ABA application on leaf discs
For cold, salt, or abscisic (ABA) treatments, 10 leaf discs from fully expanded leaves of the E. gundal line were incubated for 30 min, 2 h, or 24 h at 4 °C, or in 200 mM NaCl or 100 µM ABA. Leaf discs were incubated for similar periods in distilled water as a control for cold and salt treatments, or in dimethylsulphoxide (DMSO) as a control for ABA application. For gene expression studies, total RNA was extracted from all the tested discs.
Real time RT–PCR
The total RNA was extracted from E. gundal leaves using the SV Total RNA Isolation System (Promega, France). Using SuperScript II and random primers (Invitrogen, France), cDNAs were produced according to the manufacturer's instructions. The EguCBF1-specific primers were designated (Table 2) using the Primer Express software (version 2.0, Applied Biosystems, France). The PCRs were performed in 20 µl of 2x SYBR Green Master mix (Applied Biosystems), with 10 ng of cDNA and 300 nM of each primer. Three replicates of each PCR were run in an ABI PRISM 7900HT Sequence Detection System (Applied Biosciences, France) using a programme including a first step (50 °C/2 min and 95 °C/10 min) followed by 40 cycles (95 °C/15 s and 60 °C/1 min). The non-specific products could be detected after the end of the amplification programme, when the PCR assays were submitted to a temperature ramp in order to create the dissociation curve, measured in terms of the changes in fluorescence as a function of temperature. The dissociation programme was 95 °C /15 s, 60 °C /15 s followed by 20 min of slow ramp from 60 to 95 °C. Specific primers for 18S RNA were used as the internal control for the normalization of the RNA steady-state level, and the relative changes in gene expression were quantified using the 2–
Ct method (Livak and Schmittgen, 2001). The results of EguCBF1 relative transcript abundance are presented as a mean value of the three assay replicates compared with the mean of the three control values (leaves from control plants).
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Results |
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Isolation and structural characterization of EguCBF1a and EguCBF1b
Two CBF-like genes were isolated from E. gunnii, one through a PCR-based approach and the other from a cDNA library (unpublished data). The full-length ORF sequences were designated EguCBF1a and EguCBF1b (DQ241820 [GenBank] and DQ241821 [GenBank] ) due to their strong similarity (BLAST analysis) to other known CBF1 genes. The two genes do not exhibit any introns and encode two putative ORFs (of 220 and 223 amino acids, respectively) which bear a close resemblance to each other (82% identical).
The predicted molecular masses are 24.78 kDa for EguCBF1a and 25.13 kDa for EguCBF1b, and the estimated isoelectric points are 7.24 and 5.72, respectively. A database search revealed that the deduced amino acid sequences of EguCBF1A and B proteins contain the conserved DNA-binding domain of 60 amino acids which is characteristic of the AP2 family of plant DNA-binding proteins (underlined in Fig. 2). In addition, the two genes exhibit the CBF signature sequences ETRHP and DSAWR found upstream and downstream of the AP2 motif, and the basic residue region ‘PKKP\RAGRKKFR’ located on the N-terminal domain which might function as a nuclear localization signal (NLS).
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The amino acid sequence alignments used for comparison with other CBF proteins showed that EguCBF1A shares 67% similarity with LeCBF1 (AY034473 [GenBank] ) from tomato and 60% similarity with AtCBF1 (AY667247 [GenBank] ) from Arabidopsis; EguCBF1B shares 66% similarity with CaCBF1 (AY368483 [GenBank] ) from Capsicum and 53% similarity with AtCBF1. Based on the sequence divergence in the conserved domains of these CBF/DREB sequences, the phylogenetic tree analysis defines three main groups (Fig. 3). As expected, Eucalyptus genes belong to the group consisting of Capsicum, Lycopersicon, Gossypium, and Prunus, which represents one of the two dicotyledon groups, the second of which contains Arabidopsis.
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Promoter sequence analysis
The promoter sequence from the EguCBF1a DNA fragment contained several predictive cis-acting elements presumed to be involved in transcriptional regulation. The typical TATA box sequence was located about –30 bp from the transcription initiation point (Fig. 4). In addition, motifs related to MYB (five sites) and MYC (eight sites) recognition sites, and three ABRE cis-elements (ABA-responsive element) were detected on the EguCBF1a promoter sequence.
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DNA gel blotting
To determine the EguCBF copy number in the Eucalyptus genome, DNA was digested by EcoRI, BamHI, or HindIII which did not cut, or by EcoRV cutting once within the EguCBF1a and b sequences. After blotting, the filter was successively hybridized to two labelled probes containing the CBF well-conserved coding region or the EguCBF1-specific sequence from the 3'-terminal region. The profiles obtained with the two probes were found to be strictly identical, indicating that their hybridization is equally efficient in these experimental conditions, and providing confirmation of Southern data. The resulting complex hybridization patterns (shown only for the full-length ORF, Fig. 5) suggest that at least five EguCBF-related genes are present in the Eucalyptus genome. The EcoRI and BamHI profiles presented some similarities, in particular for the shortest band (1 kb), which was unexpected given the EguCBF1a and b restriction maps. This may result from the digestion of another EguCBF family member.
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The EguCBF1a- and b-encoded proteins are targeted to the nucleus
The subcellular localization of EguCBF1 proteins was studied using a gene fusion with the coding sequence of the GFP associated with the EguCBF1a and b coding sequences under the control of the CaMV 35S promoter. Plasmids bearing either the GFP gene alone, or the EguCBF1a::GFP and EguCBF1b::gene fusions were transiently expressed in Eucalyptus protoplasts. Fluorescence microscopy analysis associated with image overlay techniques demonstrated that control cells transformed with GFP alone displayed fluorescence spread throughout the cytoplasm, in accordance with the expected cytosolic localization of the GFP proteins (Fig. 6a). In contrast, both the EguCBF1a::GFP (Fig. 6b) and EguCBF1b::GFP (Fig. 6c) fusion proteins were localized exclusively in the nucleus, indicating that both EguCBF1a and b were fully able to redirect the GFP from the cytosol to the nucleus.
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Comparative gene expression analysis of EguCBF1a and EguCBF1b in response to various environmental conditions
For the two aligned genes, the expression was quantified by real-time RT–PCR after cold shocks to plants, or leaf disc treatments representing various abiotic conditions.
First, the cold response was evaluated on whole leaves from plants grown in controlled cold conditions. The results provided another highly significant similarity to the known CBF1: EguCBF1a and b were quickly (15 min) and strongly (up to 500 times) induced by low temperature (Figs 7, 9, 10). After exposure at 4 °C in the dark, the transcript accumulation of EguCBF1a and b reached a maximal level after 2–5 h (Fig. 7). Whereas at 24 h EguCBF1a induction declined, it was still strong for EguCBF1b. The two genes very clearly showed different levels of cold induction, EguCBF1a mRNA accumulating up to twice as much as EguCBF1b at the maximum level of induction. Interestingly, this differential pattern is the opposite for the earliest time-courses (15 and 30 min) as well as for the latest point (24 h).
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Surprisingly, light did not amplify the EguCBF1 response to cold. On the contrary, it exhibited a negative effect on induction (Fig. 7A, B). Although detectable at 4 °C in the light, EguCBF1a and b induction was 3–9 times lower compared with dark conditions, with a similar time-course of up-regulation for both conditions. As a control, the light influence on EguCBF1a and b expression in the absence of cold was also evaluated by comparison between plants cultured for 2 h at 22 °C in light or dark conditions. The slight fold change in the RNA level (2.85) observed in the dark compared with the light condition for the EguCBF1b transcript, and not shown for EguCBF1a, cannot be considered as significant compared with the ratios (up to 500) obtained after cold exposure. In addition, the importance of the time of day at which the plants were exposed to cold was checked, since this accumulation was reported in Arabidopsis to be regulated by the circadian clock (Fowler et al., 2005). Less than 11% variation (454/509-fold change) was detected over 6 h on EguCBF1a and b induction, indicating that on whole plants under cold conditions, this parameter does not significantly interfere with the other factors under study, thus allowing any induction change above 11% to be taken into account.
The effect of the magnitude of the cold shock on gene expression was then determined on the same plant material after a 2 h exposure in dark conditions (Fig. 8). For the two genes, a decreasing positive temperature resulted in higher accumulation of EguCBF1 transcripts with a dramatic increase (x25) below 8 °C and a maximum at 0 °C. At 4 °C, the same result as in the previous experiment (Fig. 7) was obtained for the two genes, showing the reproducibility of the measurements. For a freezing temperature (–4 °C), the EguCBF1 induction dropped to a very low, although still significant ratio. This decrease could be partly explained by the stress symptoms which the plants exhibited after this treatment.
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In a particularly striking result, when the two genes were compared, they were found to respond similarly to 8 °C, but above this temperature, EguCBF1b was the more highly induced, whereas EguCBF1a transcripts accumulated more below 8 °C.
In order to investigate the specificity of the EguCBF1 gene response, their expression was then measured on leaf discs exposed to cold, salt, or ABA treatment. Using this simplified system which allows the rapid and controlled application of different treatments, the level of induction was found to be 
25 times lower than in the integrated system (cf. Figs 7, 8) as shown by the control exposed for 2 h at 4 °C (Fig. 9). However, the high sensitivity of the measurements meant that changes in gene expression could be reliably detected and the fold change values were considered significant when >2-fold.
As previously observed, EguCBF1a showed a stronger response after a 2 h cold exposure compared with EguCBF1b. EguCBF1a and b were also found to respond to salt stress but to a much more limited extent than the cold response (Fig. 9). While EguCBF1b did not appear to be significantly regulated by ABA under the tested conditions, the EguCBF1a transcripts accumulated after 2 h of ABA treatment. This ABA induction was delayed compared with cold or salt stress since no induction could be detected under 2 h, and the maximum induction was observed at 24 h.
Gene expression and development of freezing tolerance in two photoperiodic conditions
The evaluation of the relationship between EguCBF1 expression and the increase of frost tolerance represents one of the main issues of this study to estimate the involvement of CBF1 in Eucalyptus cold acclimation. For this purpose, EguCBF1 expression was quantified on two sets of three plants during two specific cold culture programmes resulting in a distinct cold acclimation. The programme used the same light intensity and decrease in temperature but differed in the photoperiod (SD or LD). The measurements of freezing tolerance and gene expression were performed on the leaves which are the sites of photoperiod perception.
In both photoperiod conditions, the results of freezing tolerance measured at –6 °C as well as –8 °C (Fig. 10A, B) showed a significant and gradual increase starting from the end of the first period of the cold programme (day 4=4 d at 12 °C/day and 8 °C/night). At the end of the second period (day 10=6 d at 4 °C), the increase of freezing tolerance reached 60% for the SD (Fig. 10A) and 46% for the LD programme (Fig. 10B), showing the positive influence of an SD photoperiod on the cold acclimation efficiency. The results show only a 10 d cold-culture programme because at this stage the acclimation was found to reach its maximum and no significant improvement of freezing tolerance could be observed after an additional 4 d period at 4 °C (data not shown).
The EguCBF1 expression patterns observed in these two acclimation programmes (Fig. 10C, D) provide some important information.
- Gene expression differs according to the acclimation programme: the maximum gene induction rate for the two genes was observed in SD conditions during the 4 °C period. The gene induction rate reached a 118/191-fold change for EguCBF1a and b, respectively, in an SD photoperiod and 24/30 for the same genes during the same time course (5 h) in LD conditions. A significant difference was observed in all the kinetic points, in accordance with the higher magnitude of cold hardening obtained in SD conditions.
- The intensity of the response at 4 °C during the acclimation programme is lower than the induction observed after a direct shock at the same temperature. During the initial transfer at 12 °C, the induction ratios (ranging 5- or 10-fold depending on the gene) are similar to those observed after a 2 h direct exposure at 12 °C (Fig. 8). In interesting contrast, after the transfer from the 12 °C phase to 4 °C, EguCBF1a is 10-fold less induced and EguCBF1b is 2-fold less induced than after a 2 h direct treatment at 4 °C (Fig. 8).
- The two CBF1 genes respond differently to these acclimation programmes: EguCBF1b is always more significantly induced than EguCBF1a (often around twice as much) and is the only one to exhibit a significant response at 12 °C (Fig. 10D). It is important to note that the differential response of the two EguCBF1 genes is mostly inverse for the two conditions of cold treatment since induction is usually higher for EguCBF1a after a shock while EguCBF1b is the more up-regulated during the acclimation. However, the differential behaviour observed during the acclimation is in agreement with the responses previously observed in cold shock at 4 °C (15 min, 30 min, and 24 h, Fig. 7) or 12 °C ( 2 h, Fig. 8). In total, the expression patterns of the two genes show some complementarity: EguCBF1a exhibits an efficient short-term response to abrupt and drastic cold conditions whereas EguCBF1b shows a better and longer response to more moderate or progressive temperature changes.
- The time-course of gene induction during the 10 d experiment is very unexpected. Except for the predictable main peaks corresponding to 5 h at 4 °C, induction under the best conditions (SD) is significant (a >2-fold rate) for all the kinetic points measured, including those measured several days after a temperature change (days 6, 7, and 10). In order to check whether the EguCBF1 transcripts are unusually stable or produced anew as a reaction to repeated cues, the transcription rate was more precisely quantified for the most induced gene (EguCBF1b). The time-course was studied during an SD acclimation programme and in particular 15 min before and 2 h after the thermoperiod transition (12 °C day/8 °C night). This detailed kinetics (Fig. 11) confirmed the likely occurrence of subsequent gene inductions at least every day as accumulation of transcripts is always higher after the phase transition. This temperature change (12/8 °C) combined with switching off the light could represent a daily trigger to induce EguCBF1. This result agrees with the slight negative influence on gene regulation by cold already observed for the photoperiod effect and for the 4 °C cold shock in the dark or in continuous light. However, on the 4th day of the acclimation programme, while the thermoperiod transition still had a positive influence, the induction level was significantly lower than on the previous days, suggesting a reduction in the impact of this daily trigger.
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Overall, the transcriptional response of sensitive genes such as EguCBF1s seems highly dependent both on the conditions of cold occurrence and on the associated photoperiod. Therefore, if the short exposure to 4 °C seems to be an efficient system for evaluating the different parameters of regulation quickly and qualitatively, the culture leading to acclimation that mimics natural conditions is more representative of the response in the field.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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This study presents a quantitative transcriptional analysis of two CBF-like genes in a woody plant in response to different environmental conditions. These genes exhibit all the major structural characteristics of a CBF transcription activator gene, including the absence of an intron also reported for Arabidopsis (Medina et al., 1999) or tomato (Zhang et al., 2004). In agreement with the predicted identity as a transcriptional regulator, the nuclear location of the protein was proved by the in vivo targeting experiment. Based on these characteristics and the highest level of homology to CBF1, the two genes were designated EguCBF1a and EguCBF1b. These new genes are paralogues because they are clearly different; the polymorphism between the two sequences is even higher than between the well-known Arabidopsis members CBF1–CBF3.
Several promoter cis-acting elements common to the known CBF genes were predicted in EguCBF1a, in particular the MYC recognition sequences that would be involved in CBF transcriptional activation by cold (Chinnusamy et al., 2003). This cold regulation was evidenced by the expression analysis showing a strong and fast response to cold, typical of a CBF gene. Induction of EguCBF1a by cold is 10 times higher than the accumulation of transcripts measured by the same method for AtCBF1–2 in similar cold conditions (Rohde et al., 2004). According to the hypothesis of Chinnusamy et al. (2003), this higher induction level could be related to the number of MYC elements, eight for EguCBF1a and only one for AtCBF1–2 (Shinwari et al., 1998). Moreover, the five MYB transcription factor recognition sites predicted in EguCBF1a, as well as AtCBF1–2 (four or two sites, respectively), could be involved in the CBF activation by cold, as suggested by the same authors (Chinnusamy et al., 2003).
Overall, since CBF transcript quantifications have rarely been reported, it is difficult to compare the EguCBF1 induction levels with data from the literature. The comparative transcriptional analysis of two CBF members in multiple conditions as described in this report was only made possible by the use of the powerful real-time RT–PCR technique. Benefiting from the quantitative and highly sensitive measurements of the transcriptional regulation and the possible multiplication of assays, the presented data have proved highly significant and reproducible. They underline the importance of applying such a method to further studies on a weakly expressed transcriptional activator gene such as CBF.
In addition to these MYC and MYB recognition sites, three ABRE cis-elements were predicted in the EguCBF1a promoter in accordance with a detected response to ABA treatment. In Arabidopsis, a potential ABRE cis-acting element was predicted in AtCBF1–3 promoters (Busk and Pages, 1998; Medina et al., 1999), but the situation still remains unclear concerning the participation of this hormone in CBF regulation (Medina et al., 1999; Knight et al., 2004). Surprisingly, in addition to cold, the EguCBF1 response to salt was significant even though it was impossible to compare the two regulations quantitatively in the absence of any optimization of the experimental conditions for NaCl. This response is rather uncommon since AtCBF1–3 as well as LeCBF1 are usually described to have cold-specific responses (Medina et al., 1999; Zhang et al., 2004). However, during a global survey through microarray analysis of the transcriptome changes in response to different environmental cues, Kreps et al. (2002) observed a strong induction by cold and a slight but significant up-regulation by salt or mannitol for AtCBF1. In addition, Choi et al. (2002), by using real-time RT–PCR, were able to reveal a small transient induction of HvCBF3 after ABA treatment, although nothing was detected by standard reverse transcription analysis (semi-quantitative), which confirms that these data regarding response specificity may vary according to the method of measurement. It is therefore difficult to draw any conclusions about potential differences or similarities for the specificity of response to environmental stimuli in the comparison of CBF genes from Eucalyptus and other species.
However, clearer differential features were observed regarding the effect of light on CBF regulation by cold. In contrast to the data on AtCBF (Kim et al., 2002), darkness during cold shock, as well as the application of an SD photoperiod during the acclimation programme, has a strong positive effect on EguCBF1 expression. In neither case did light act as an independent and adequate signal. Instead, it acted by quantitatively modulating the EguCBF1 gene response, which is primarily regulated by low temperature. This predominance of temperature over light has already been reported for growth and a number of other physiological responses in Eucalyptus when 12 different species, grown under different combinations of temperature and photoperiod, showed greater response to temperature than to photoperiod (Paton, 1978). The data presented on the photoperiodic response of Eucalyptus are also fully in agreement with the literature for adaptive traits associated with overwintering on many perennial woody plants (Thomas and Vince-Prue, 1997; Welling et al., 2002).
The involvement of light in fine-tuning plant molecular responses to cold has often been described and, as in Eucalyptus, the positive effect of dark or an SD photoperiod on the regulation of cold-responsive genes has been reported for other plant species including barley (Grossi et al., 1998; Fowler et al., 2001) and poplar (Zhu and Coleman, 2001). The very fast effect of darkness on the EguCBF1 cold response (already significant after 15 min) as well as the influence of photoperiod on gene expression strongly suggests that a photoreceptor is involved rather than the photosynthetic electron chain observed in this regulation. In Arabidopsis, phytochrome B appears to act primarily as a light signalling mediator for CBF regulation (Kim et al., 2002) while in Populus, phytochrome A appears to be the main intermediary for dehydrin gene regulation (Zhu and Coleman, 2001).
The contrasting response to light between Arabidopsis and Eucalyptus suggests that although they exhibit many similarities with AtCBF genes, the EguCBF genes differ significantly both structurally and in their regulation. These differences may be related to the very characteristic biology and physiology of this persistent woody plant compared with an annual species such as Arabidopsis, which survives winters through seed storage. These first data on the expressional analysis of CBF genes from a forestry species highlight the utility of in-depth study of this predominant cold response pathway. Such a study needs to be carried out on a complex perennial model like the present one in order to reveal the molecular mechanisms underlying many of its unique developmental features.
One of the most useful results of this study is the apparent relationship between the development of freezing tolerance and the induction level of the two EguCBF1 genes highlighted by the comparison of the two acclimation programmes. The involvement of CBF genes in cold hardening is now well documented, most commonly through the genetic modulation of gene expression on model species. The involvement of a CBF gene from a woody plant (sweet cherry) in freezing tolerance was demonstrated for the first time by Kitashiba et al. (2004) by PaCBF expression in Arabidopsis. However, such heterologous approaches remain limited and beg the question of the efficacy of the CBF pathway in the species under study. For example, due in particular to a limited regulon or reduced induction by cold, the LeCBF gene did not work for tomato cold acclimation whereas it was found to enhance freezing tolerance when transferred to Arabidopsis (Zhang et al., 2004). In a broader range of plant species, the comparison of cold-tolerant or cold-sensitive genotypes is now an emerging approach to investigate similarities and differences in the CBF cold response pathway. The definition of the two acclimation experiments on Eucalyptus and the use of a very efficient and sensitive transcript level measurement provided a first approach to this gene regulation in a woody plant associated with the level of frost tolerance. The extremely strong and rapid EguCBF1 induction by cold, in agreement with the tolerance level in leaves, suggests the involvement of the two genes in Eucalyptus cold hardening. This must be confirmed by genetic modulation in an available homologous system (Tournier et al., 2003; Valério et al., 2003).
Among the main data from this quantitative expressional analysis, the two EguCBF1 genes were found to be differentially induced depending on the speed and intensity of the temperature drop. Such a distinct regulation was also established for the AtCBF members, for example when AtCBF2 induction was found to be delayed compared with AtCBF and AtCBF3 (Novillo et al., 2004). When the three AtCBF genes were found to show redundant functional activity in AtCBF1–3-overexpressing plants, Gilmour et al. (2004) raised the question of whether they are functionally equivalent in planta. As a first step towards an answer, the presented data on Eucalyptus strongly suggest that the two studied EguCBF members may be regulated in the field in a complementary manner.
The present study also provides a range of information about the conditions of cold treatments affecting the accumulation of transcripts. In agreement with the findings on Arabidopsis (Zarka et al., 2003), the intensity of the cold obviously proved to be critical since, during a direct cold exposure in a positive temperature range, lower temperature results in higher induction. However, the EguCBF1 transcript accumulation level was not as proportional to the temperature decrease as observed for AtCBF and, above all, a negative temperature only results in a low induction in Eucalyptus, whereas it is the best stimulus for Arabidopsis. These data are consistent with the evidence about lower temperature applied for acclimating Arabidopsis (4 °C), which was too stressful to harden Eucalyptus directly. Together they indicate the best compromise between an efficient stimulus and moderate stress ranges at higher temperatures for this woody plant. More importantly, the rapidity or progression of cold exposure proved to be relevant to the EguCBF1 response since, for the same temperature (4 °C), the induction level during the two-step acclimation programme was lower than after a direct shock. This observation suggests that the cold response of EguCBF1 is not only limited to a direct monitoring of absolute temperature but also takes the temperature difference into account. It could also reflect the desensitization phenomenon described by Zarka et al. (2003) for AtCBF who observed a loss of cold response after 14 d at 4 °C. This apparent loss of response, also shown by the study of the detailed kinetics at 12 °C, was already detected in our hands for cold acclimation (unpublished data). Therefore, like Arabidopsis, Eucalyptus cells probably keep a memory of chilling exposure by adjusting the CBF response in particular. The dynamism of this EguCBF1 response over numerous temperature and light changes which occur permanently in natural conditions was also shown by the study of EguCBF1a and b expression over 10 d. As expected, subsequent decreases of temperature were found to lead to corresponding increases in transcript level. However, considering the CBF RNA half-life, 7.5 min for Arabidopsis (Zarka et al., 2003), the basic induction level remained surprisingly high for several days, even at 4 °C in the absence of any new temperature stimulus. In addition, new CBF induction peaks were observed every day at each photoperiod/thermoperiod transition during the 12 °C culture step. Such a long-term regulation of a CBF1 gene was only recently suggested for the first time by Hannah et al. (2005) in an overview of gene regulation during cold acclimation in Arabidopsis. The authors mentioned that although AtCBF1 and AtCBF3 were supposed to be only transiently expressed because they were tightly negatively controlled by AtCBF2 (Novillo et al., 2004), the data indicated the likelihood that they were up-regulated in the long term (>48 h). In addition, the same authors found that 12 other short-term responsive transcription factor genes continue to be induced during a long-term cold acclimation. The present work provides a possible explanation for this apparently ‘permanent’ long-term induction with the occurrence of repeated inductions in response to environmental changes (temperature, light, etc.). Altogether, the results underline how complex and dynamic this gene regulation is. It is affected not only by various environmental factors but also by the history of the cells, which have an apparent memory for cold.
An original feature of this study is the monitoring of CBF expression in controlled conditions closely corresponding to the natural environment (day and night temperatures, light intensity, and photoperiod) and compatible with cold acclimation and long-term plant survival. The differences observed in the CBF response between the ‘shock experiments’ and the ‘acclimation experiments’ confirmed that this approach is crucial for assessing the real involvement of CBF in cold tolerance and its regulation during autumn and winter. These interesting new data on the transcription factor gene suggest that, in the field, it is permanently subjected to positive and negative regulation and can be quickly induced in response to each temperature decrease. However, due to the memory effect, this response would show a higher amplitude in non-acclimated conditions than in an acclimated plant state (late autumn and winter).
In the near future, the isolation of at least three other members of the EguCBF family, coupled with a quantitative analysis of their transcriptional pattern both in controlled conditions and in the field, should provide an overview of the CBF regulation during overwintering of this perennial woody plant.
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Acknowledgements |
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We would like to thank AFOCEL for providing the Eucalyptus cuttings. Thanks to Helene San Clemente for bioinformatic analyses and Yves Martinez for confocal microscopy analysis. This work was supported by the ‘Midi Pyrénées French Council’, TEMBEC SA R&D KRAFT (St GAUDENS, France), and European funds for development (FEDER).
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Footnotes |
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* Present address: Biotechnology Research Center, Forest Resources and Environmental Science, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931-1295, USA
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Abbreviations |
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ABA, abscisic acid; ABRE, ABA-responsive element; AFOCEL, Association Forêt Cellulose; CaMV, cauliflower mosaic virus; CRT/DRE, C-repeat/dehydration-responsive element; EST, expressed sequence tag; GFP, green fluorescent protein; LD, long day; ORF, open reading frame; RT–PCR, reverse transcription–polymerase chain reaction; SD, short day.
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