Journal of Experimental Botany, Vol. 52, No. 360, pp. 1545-1554,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
Accumulation of plastid lipid-associated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species
1 Génétique Moléculaire des Plantes, Université J Fourier and CNRS (UMR 5575), BP 53, F-38041 Grenoble, cedex 9, France
2 CEA/Cadarache, DSV, DEVM, Laboratoire d'Ecophysiologie de la Photosynthèse, F-13108 Saint-Paul-lez-Durance cedex, France
Received 2 October 2000; Accepted 13 March 2001
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Abstract |
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Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are known to accumulate in fibrillar-type chromoplasts such as those of ripening pepper fruit, and in leaf chloroplasts from Solanaceae plants under abiotic stress conditions. It is shown here that treatments generating active oxygen species (high light combined with low temperature, gamma irradiation or methyl viologen treatment) result in potato CDSP34 gene induction and protein accumulation in leaves. Using transgenic tomato plants containing the pepper fibrillin promoter, a significant increase in promoter activity in leaves subjected to biotic stress, namely bacterial infections, was observed. In WT, a higher level of the endogenous fibrillin/CDSP34 protein is also observed after infection by E. chrysanthemi strain 3739. In addition to stress-related induction, a progressive increase in the fibrillin promoter activity is noticed during ageing in various tomato photosynthetic tissues and this increase correlates with a higher abundance of the endogenous protein in WT leaves. It is proposed that a mechanism related to oxidative events plays an essential role in the regulation of fibrillin/CDSP34 genes during stress and also during development. Using a biolistic transient expression assay, the pepper fibrillin promoter is found to be active in various dicot species, but not in monocots. Further, substantially increased levels of fibrillin/ CDSP34 proteins are shown in various dicotyledonous and monocotyledonous plants in response to water deficit.
Key words: Abiotic and biotic stress, ageing, CDSP34/fibrillin accumulation, oxidative stress, Solanaceae.
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Introduction |
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Plant fibrillins, also termed plastid lipid-associated proteins or ChrC, are nuclear-encoded plastid proteins up-regulated during chromoplast differentiation in certain fruits (Deruère et al., 1994
) and flowers (Vishnevetsky et al., 1996). In pepper, fibrillin is necessary for the assembly of the carotenoid-storing fibrils in chromoplasts during fruit ripening (Deruère et al., 1994). Recently, the protein was also found to accumulate in drought-stressed pepper leaves (Chen et al., 1998). This has been further evidenced by the cloning of a cDNA encoding a potato chloroplast protein highly homologous to pepper fibrillin (Gillet et al., 1998) and previously characterized as a water stress protein termed CDSP34 (Chloroplast Drought Stress Protein of 34 kDa, Pruvot et al., 1996a). The protein, localized in the extrinsic fraction of stromal lamellae thylakoids (Pruvot et al., 1996a; Eymery and Rey, 1999), was shown to accumulate under different environmental constraints such as low temperature or high light (Pruvot et al., 1996b; Gillet et al., 1998). Fibrillin-related proteins have also been found associated with plastid lipid-rich structures in other species such as pea (Kessler et al., 1999) and Brassicaceae (Ting et al., 1998; Hernández-Pinzón et al., 1999). Moreover, related sequences have been revealed by systematic sequencing projects in Arabidopsis thaliana (Genbank AL021712, AF075598) and Zea mays (Genbank AA979828) suggesting the widespread occurrence of fibrillin proteins in plants.
In non-photosynthetic plastids, fibrillin proteins are most likely involved in the storage of hydrophobic compounds such as carotenoids (Deruère et al., 1994) and lipids (Ting et al., 1998). The function of the protein within chloroplastic thylakoid membranes remains unclear. Based on an antisense approach, it has been proposed that the potato protein was involved in the modulation of photosynthetic efficiency (Monte et al., 1999). On the other hand, it has been suggested that the association of fibrillin/CDSP34 with lipids could be involved in the structural stabilization of thylakoid membranes upon environmental constraints (Gillet et al., 1998; Chen et al., 1998). Studies on fibrillin over-expressing tobacco plants indicated that environmental stress-induced association of the pepper protein with thylakoids and showed that transgenics exhibited increased growth and modified development under high light conditions (Rey et al., 2000). In summary, the available data suggest that fibrillin/CDSP34 proteins fulfil important functions in leaves potentially linked to the protection of thylakoid membranes and plant growth upon stress.
Expression of fibrillin/CDSP34 genes has been described as occurring in response to abiotic stress or during development of diverse plant organs. In tobacco and tomato leaves, the pepper fibrillin promoter is induced during stress by photo-oxidative events involving active oxygen species (AOS) (Chen et al., 1998; Manac'h and Kuntz, 1999). Based on experiments showing enhanced expression of the CDSP34 gene in potato plants subjected to high light, the involvement of oxidative events in the gene induction in leaves upon environmental stress has also been suggested (Gillet et al., 1998). Indeed, under abiotic constraints, the photosynthetic apparatus is a major site of AOS formation (Asada, 1994; Smirnoff, 1993) that can result in damage such as lipid peroxidation (Mishra and Singhal, 1992). In addition, the pepper fibrillin promoter also appears to be up-regulated by mechanisms involving redox changes during ripening of tomato fruit (Kuntz et al., 1998). On the other hand, different developmental factors have been reported to regulate the expression of fibrillin genes in plants. In developing cucumber flowers, the expression of the ChrC gene encoding a fibrillin homologue is rapidly up-regulated by gibberellic acid and down-regulated by abscisic acid and ethylene (Vishnevetsky et al., 1999a). a developmental control for the expression of the related gene in leaves of S. demissum plants during tuberization has also been proposed (Monte et al., 1999).
To further these studies, it was found necessary to gain a better understanding of the expression of fibrillin/ CDSP34-related genes in leaves in relation either to stress or development. The induction of fibrillin/CDSP34 genes in response to oxidative and biotic stress conditions and also during ageing in leaves of Solanaceae is reported. It is proposed that a pathway related to photo-oxidative events plays an essential function in the up-regulation of the fibrillin/CDSP34 gene in leaves. Drought-induced accumulation of fibrillin/CDSP34-related proteins in leaves of various species including a monocotyledonous plant is also shown.
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Materials and methods |
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Plant material, growth conditions and experimental treatments
Tomato (Lycopersicon esculentum L., cv. Ailsa Craig) wild-type plants and the transgenic lines carrying the promoter of the pepper fibrillin gene in fusion with the GUS coding region (Kuntz et al., 1998
; for details on constructs see below the DNA construct paragraph) were grown as described (Manac'h and Kuntz, 1999). Previously described lines 11–16 (Kuntz et al., 1998) which were found to show consistent expression of the reporter gene in leaves (Manac'h and Kuntz, 1999) were used. Bell pepper plants (Capsicum annuum L., cv. Yolo Wonder) were grown under the same conditions as tomato plants.
Potato plants (Solanum tuberosum L., cv. Haig), originating from in vitro plantlets, were grown on compost in a phytotron (12 h photoperiod, 300 µmol m-2 s-1 unless otherwise stated) as described earlier (Pruvot et al., 1996b). Oxidative treatments were performed with 3-week-old potato plants. High light treatment (1200 µmol m-2 s-1) and low temperature (8 °C) were applied for 3–6 d. Gamma irradiation (100 Grays) was performed using a source of cobalt 60 delivering 0.659 Grays min-1. Methyl viologen treatment was performed by spraying each plant with 10 ml of a solution of 10 µM methyl viologen (Sigma, St Louis, USA) in 0.25% Tween 20. For experiments using detached leaves, petioles were cut with a razor blade and soaked for 3 or 5 h in water containing 1 mM or 10 mM H2O2, 1 µM methyl viologen or 5 mM GSSG (oxidized glutathione) under light conditions (20 µmol m-2 s-1).
Craterostigma plantagineum Hochst. plants were kindly provided by Dr D Gaff (Monash University, Clayton, Australia). Maize (Z. mays L.) and barley (Hordeum vulgare L., cv. Plaisant) seeds were purchased from a local seed supply company. Progressive water deficit was induced in different plant species grown in a phytotron by withholding water for 8–12 d. Relative water content, RWC (fresh weight–dry weight)/(water saturated weight–dry weight), was determined on leaf pieces as described previously (Pruvot et al., 1996b).
Biotic stress
Leaves from the above-mentioned fibrillin promoter-containing tomato lines were vacuum-infiltrated in the following elicitor solution (100 nM): oligomannosyl N-glycans (a mixture of Man4-9GlcNAc), a xylomannosyl N-glycan (Man3(Xyl)GlcNAc(Fuc)GlcNAc) and chitopentaose. The latter elicitor binds to a receptor on the tomato cell membrane (Baureithel et al., 1994) and induces various cellular responses, such as pH changes, protein phosphorylation and gene activation (Felix et al., 1993) thus mimicking a pathogen attack. The free N-glycans used here were chosen since they had been reported to stimulate tomato fruit ripening (Priem et al., 1993). Man3(Xyl)GlcNAc(Fuc)GlcNAc was also described as a growth factor (Priem et al., 1990). Treated leaves were incubated for 24 h with a water supply as described earlier (Manac'h and Kuntz, 1999). For pathogen infections, droplets (30 µl in total) from an overnight culture grown in LB medium were deposited on top of a detached leaf from the transgenic lines. As a control, LB droplets were deposited on top of leaves from the same transgenic lines. Leaves were then incubated for 24 h in light or in darkness for GUS activity measurements and for 55 h in light for Western experiments. Pathogens used were Erwinia chrysanthemi strains EC16, 3739 and ENA49, and E. carotovora with the subspecies betavasculorum, carotovora and odorifera. All Erwinia strains were kindly provided by N Cotte-Pattat, INSA, Lyon.
Extraction of leaf proteins, electrophoresis and immunoblot analysis
After blending leaflet samples (1 g) in liquid N2, the powder was resuspended in 50 mM Tris-HCl, pH 8.0, 50 mM ß-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride, and centrifuged (10 000 g, 4 °C, 10 min). The pellet, containing thylakoids, was resuspended in 50 mM TRIS-HCl pH 8.0, 1% SDS, agitated for 30 min at 4 °C, then centrifuged (10 000 g, 4 °C, 15 min). Membrane proteins were precipitated at -20 °C by the addition of 4 vols of acetone to the supernatant. Protein content was determined using a modified Lowry method (Sigma).
Electrophoresis (Laemmli, 1970) was performed in 12% (w/v) acrylamide gels. Proteins (20 µg per lane) were separated by SDS-PAGE and were electroblotted onto 0.45 µm nitrocellulose (Schleicher and Schuell, Dassel, Germany). Nitocellulose membranes were stained with Ponceau red to ensure that equal protein amounts had been loaded. Western blot analysis was performed as described previously (Gillet et al., 1998). The serum raised against the N-terminal part of potato CDSP34 was prepared as reported earlier (Pruvot et al., 1996a) and was used diluted (1 : 1500). The serum raised against the whole mature CDSP34 was obtained as follows. The potato CDSP34 cDNA was cloned in PQE31 (Qiagen) and the plasmid transferred into the E. coli strain M15REP4. A fusion protein composed of 6 His at the N-terminal end of the mature CDSP34 was produced and purified on a Ni affinity column (Ni-NTA Agarose, Qiagen). A serum was raised against the recombinant protein in a rabbit and was used diluted (1 : 4000). Bound antibodies were detected using an anti-rabbit immunoglobulin-G alkaline phosphatase conjugate (Roche Diagnostics, Mannheim, Germany).
RNA isolation and RNA gel blot analysis
Leaf samples (1 g) were collected and immediately frozen in liquid N2. Total leaf RNA was extracted and size separated in formaldehyde gels (20 µg per lane) as described earlier (Rey et al., 1998). Gels were stained with ethidium bromide to ensure that equal amounts of RNA had been loaded. After blotting onto Biodyne B (Pall Gelman Sciences, Ann Arbor, USA), hybridization to the full length CDSP34 cDNA probe (random-primed labelled with [
-32P]dATP) was carried out in 7% PEG-8000, 10% SDS and 100 µg ml-1 salmon sperm DNA at 65 °C for 16 h (Broin et al., 2000). Washing and autoradiography were as described previously (Gillet et al., 1998).
DNA constructs and transient transformation of leaf-tissue
A fusion construct consisting of the fibrillin promoter region (a 2296 bp DNA fragment upstream of the translation initiation codon) fused to the GUS reporter gene and the nopaline synthase terminator (Chen et al., 1998), also present in the transgenic lines, has been used for transient assays. Alternatively the capsanthin-capsorubin synthase (CCS;2310 bp) or CaMV-35S promoters inserted in otherwise identical constructs have been used.
Preparation of 1 µm diameter gold particles (60 mg gold sterilized with ethanol and resuspended in 1 ml sterile water) and coating of DNA was performed as described previously (Sanford et al., 1993). Briefly, 50 µl of particle suspension were mixed, while vortexing, with 5 µl plasmid DNA (1 µg µl-1), 50 µl CaCl2 (2.5 M) and 20 µl 0.1 M spermidine. After 10 min of vortexing, particles were pelleted by pulse centrifugation, washed with 250 µl absolute ethanol and finally resuspended in 60 µl absolute ethanol. A 6 µl aliquot of particle/DNA mixture was used for each bombardment in a PDS-1000/He Particle Delivery System (Bio-Rad). Chamber vacuum level was 84.6 kPa and the helium pulse was 7.58 MPa. For bombardments, leaves were placed upside down on 55 mm Petri dishes containing 0.6% agar and MS salts (Sigma, St Louis, USA) supplemented with 0.5%sucrose. Within 5 min after bombardment, leaves on Petri dishes were incubated for 24 h at 25 °C, with 16 h light (light intensity was approximately 170 µmol m-2 s-1) and 8 h darkness. Bombardments were performed at least three times for each species and for each DNA construct.
GUS assays
Fluorometric assays, preparation of leaf extracts and enzymatic reactions were performed as described (Kuntz et al., 1998; Manac'h and Kuntz, 1999) except that a Turner Designs TD-360 Mini-Fluorometer was used. Since GUS activity is at slightly different quantitative levels in the transgenic lines used (most likely due to position effects), the data presented in a given figure are mean values for one line only. However, assays were routinely performed using at least three different lines and lead to similar qualitative results. For a given set of experiments, replicates were performed using the same plant but with separate extractions: one foliole forming a tomato leaf was used for two separate extractions (on each side of the main vein). GUS activity is expressed as pmol methylumbelliferone (MU) formed min-1 mg-1 protein in the extract. Protein content was determined using the Bio-Rad protein assay kit based on the Bradford method. Histochemical staining to visualize GUS activity was performed essentially as described including ethanol decoloration (Jefferson, 1987).
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Results |
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Oxidative stress leads to accumulation of CDSP34 mRNA and protein in potato leaves
Available data indicate that photo-oxidative mechanisms participate in the transcriptional activation of the pepper fibrillin gene in tobacco and tomato leaves (Chen et al., 1998
; Manac'h and Kuntz, 1999). Similar mechanisms have been suggested to participate in the expression of the potato CDSP34 gene upon environmental constraints (Gillet et al., 1998). To establish whether the regulatory mechanisms demonstrated for the pepper gene are involved in the induction of the potato gene, CDSP34 expression was investigated under conditions known to generate active oxygen species and photo-oxidative stress in leaves, i.e. (i) high light combined with low temperature conditions, leading to an inhibition of CO2 assimilation due to reduced Calvin cycle activity and an excess of light, both of which favour generation of singlet oxygen and superoxide anions in chloroplasts (Cornic, 1994), (ii) ionizing radiation, which provokes the formation of hydroxyl radicals (OH·) and hydrogen peroxide (H2O2) from water (Levitt, 1980), (iii) spraying an electron acceptor at the PSI site, methyl viologen, which reduces O2 and generates superoxide anions (O2·-) in the chloroplast (Babbs et al., 1989). Potato plants subjected to these treatments exhibited typical features of oxidative damage, i.e. anthocyanin accumulation, bleaching and some necrosis in leaves (data not shown). All treatments led to enhanced expression of CDSP34 gene in leaves, but with different response kinetics (Fig. 1). Under high light/low temperature conditions, CDSP34 mRNA levels continuously increased during the 6 d treatment (Fig. 1A). In comparison, a much higher abundance of CDSP34 protein was observed after 4 d of treatment and some decrease was noticed after 6 d (Fig. 1B). A different pattern was observed for gamma irradiation and methyl viologen treatment: CDSP34 mRNA levels increased dramatically up to 1 d while protein levels were unaffected (Fig. 1C–F). At later stages (2–3 d) of treatment, the mRNA levels decreased whereas CDSP34 protein levels increased (Fig. 1C–F). These data show that CDSP34 mRNA steady-state levels are elevated in potato leaves by signals related to photo-oxidative stress conditions. In addition, non-synchronized changes in mRNA and protein levels were noticed, particularly upon gamma irradiation and methyl viologen treatments, indicating that regulation of fibrillin/CDSP34 in response to stress is more complex than exclusively a change in mRNA levels. Similar observations have been reported concerning the response to oxidative stress of another gene encoding a chloroplastic stress protein (Broin et al., 2000).
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irradiated (100 Grays) and grown in the phytotron for 6 h (6h), 1 d (1d), 2 d (2d) or 3 d (3d). (E, F) Plants sprayed with 10 µM methyl viologen (MV) and grown in the phytotron for 6 h (6h), 1 d (1d), 2 d (2d) or 3 d (3d). (CT) control plants kept under normal growth conditions.Further short-time experiments were performed on detached potato leaves fed through the petiole with solutions containing various pro-oxidant molecules (1 or 10 mM H2O2, 1 µM methyl viologen or 5 mM GSSG). Only H2O2 was found to induce a noticeable and rapid accumulation of the CDSP34 transcript (Fig. 2
; data not shown for methyl viologen and GSSG). After 3 h, H2O2 treatment resulted in 2- and 5-fold increases in the mRNA abundance at 1 mM and 10 mM, respectively (Fig. 2). CDSP34 protein levels did not change over the experimental time-course (data not shown).
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Biotic stress also activates the pepper fibrillin promoter and induces an increase in fibrillin protein abundance in leaves
The expression of fibrillin-related genes has, until now, been reported to be induced by most abiotic stress conditions in leaves of Solanaceae (Pruvot et al., 1996b; Gillet et al., 1998). The effect of biotic stress on fibrillin/ CDSP34 expression was examined using transgenic tomato lines carrying a construct containing the promoter (2.2 kb) of the pepper fibrillin gene fused to the GUS reporter gene (hereafter referred to as fib). Tomato leaves from transgenic lines were treated with biologically active elicitors (Priem et al., 1993; Felix et al., 1993), namely oligomannosyl N-glycans, a xylomannosyl N-glycan (Man3(Xyl)GlcNAc(Fuc)GlcNAc) and chitopentaose (see Materials and methods). These compounds had little effect in young leaves (not shown) and only moderately induced the fibrillin promoter in mature leaves (Fig. 3A).
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To examine the effect of biotic stresses on fib, transgenic tomato leaves were infected with cultures of E. chrysanthemi or E. carotovora strains. As shown in Fig. 3B
, clear induction was obtained with most strains after 24 h. Infection with concomitant wounding of leaves usually resulted in higher levels of GUS activity (data not shown). When the culture of E. chrysanthemi strain 3739 (which triggered the strongest induction) was centrifuged to separate bacteria and growth medium, the strongest inductive effect was found with the supernatant (Fig. 3C, left panel), suggesting that diffusive elicitor(s) from the bacteria, accumulating in the medium, are involved in the induction. This is consistent with known data that Erwinia cultures secrete cell wall-degrading enzymes, for example, into the medium (Collmer and Keen, 1986). It should be mentioned that neither the medium alone nor the bacterial resuspension solution (1% glycerol) had an effect on fib induction (Fig. 3C, left panel). Interestingly, since in darkness there was no significant induction (Fig. 3C, right panel), the observed induction appears to be light-dependent. The abundance of the endogenous fibrillin/CDSP34 protein in leaves of WT tomato infected by E. chrysanthemi strain 3739 for 55 h in light was then examined using immunoblot analysis. As shown in Fig. 3D, a noticeably higher amount of endogenous protein was observed in infected leaves.
Developmental and tissue specific regulation of the fibrillin promoter and fibrillin protein levels
The results reported above as well as previous data (Chen et al., 1998; Gillet et al., 1998; Manac'h and Kuntz, 1999) clearly show a stress induction of fibrillin/CDSP34 genes in leaves. In other respects, an induction of the gene in Solanum demissum leaves upon tuberization was described (Monte et al., 1999), which was interpreted as a developmental regulatory mechanism involving metabolic changes. Therefore, a possible alternative regulation of fibrillin/CDSP34 genes was re-examined during development in another Solanaceae, i.e. tomato.
Using the transgenic tomato lines, expression of the pepper fibrillin promoter was first investigated at different stages of leaf development (Fig. 4A). A progressive increase in promoter activity was observed during leaf ageing, reaching a c. 8-fold increase in the oldest leaves with respect to young leaves. When cotyledons were examined, gene expression levels similar to those in young leaves were observed in young cotyledons, with a slight induction observed in older cotyledons (Fig. 4B). Similarly, higher fib gene expression levels were clearly observed in older stems (lower part of plants) versus younger stems (not shown).
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In order to investigate whether the activity of the pepper fibrillin promoter in tomato plants correlates with changes in the endogenous fibrillin/CDSP34 protein levels, immunoblot analysis was performed with protein extracts from WT leaves. First, young and oldest leaves from tomato plants upon vegetative growth (0.4 m high, see details in legend to Fig. 4C
) were investigated: in three plants, the oldest leaves exhibited a higher fibrillin/ CDSP34 protein level than the young leaves. In addition, two older flowering plants (1.3 m high, see details in legend to Fig. 4C) were analysed: a continuous increase in the fibrillin/CDSP34 amount was also observed during leaf ageing in the two plants as shown in Fig. 4C.
The pepper fibrillin promoter is active in various dicotyledonous species and fibrillin/CDSP34 proteins accumulate in both monocots and dicots upon water deficit
Up to now, the data showing the induction of fibrillin/ CDSP34 genes in leaves by stress have been exclusively obtained in Solanaceae plants. To extend these data, it was decided to analyse the activity of the pepper fibrillin promoter in a wider range of species by performing biolistic transient expression assays using the above-mentioned fib construct. As controls, two different promoters, also fused to the GUS gene, were used: firstly, the constitutive CaMV-35S promoter and secondly the promoter of the capsanthin-capsorubin synthase (CCS) gene. Activity of the latter promoter has been found in fruit tissue but not in leaves in pepper (Chen et al., 1998) and transgenic tomato (Kuntz et al., 1998) plants and, therefore, it served as a negative control in experiments using leaves. As expected, bombardment of pepper leaves with fib results in strong expression of the GUS gene but, on average, signals obtained with the constitutive 35S promoter were more intense than those with fib (Fig. 5A). Bombardment of pepper leaves with the CCS promoter-GUS construct did not result in blue staining (Fig. 5A), confirming that the signals obtained with fib are not due to non-specific activation of the GUS reporter gene. The functionality of the CCS-GUS construct was checked by bombardments of fruit tissue, where in vitro GUS activity was measured (data not shown).
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Using the same transient assay, it was shown that the pepper fibrillin promoter is also active in potato leaves (Fig. 5B
). Although higher background of endogenous GUS activities were obtained when using these assays on tomato or Arabidopsis leaves, signals resulting from the activity of the fibrillin promoter can be observed in transformed cells from these species, as shown for the representative experiments presented in Fig. 5C and D. However, no signals could be observed in the two monocots tested namely maize (Fig. 5E) and barley (not shown). As a control, the 35S promoter was shown to be active in maize leaves. These data were extended by investigating the abundance of fibrillin/CDSP34 related proteins in the leaves of some species outside the Solanaceae family, namely A. thaliana, C. plantagineum (a dessication-tolerant plant; Bartels et al., 1990) and a monocotyledon, barley. Using a serum raised against a recombinant potato CDSP34 protein produced in E. coli, immunoblot analysis revealed low levels of fibrillin/CDSP34-related proteins in well-watered plants of all species (leaf RWC around 90–95%). Under water deficit conditions (leaf RWC around 50–65%), the abundance of these proteins was noticeably higher in Solanaceae as well as in the three other species including the monocotyledon (Fig. 6). Note that a strong increase in protein amount was observed in C. plantagineum (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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It is reported here that the accumulation of fibrillin/ CDSP34-related proteins upon stress conditions, such as water deficit, is a phenomenon which occurs in leaves of various dicot species and of at least one monocot. Further, it is also shown that fibrillin/CDSP34 genes are induced, at least in Solanaceae, by biotic stress, ageing and oxidative stress.
Substantially higher amounts of CSDP34 mRNA and protein were observed in potato plants subjected to various treatments known to trigger photo-oxidative stress, i.e. high light combined with low temperature, ionizing radiation and methyl viologen spraying. Therefore, taken with the data of other authors (Chen et al., 1998; Manac'h and Kuntz, 1999) who reported a transcriptional activation of the fibrillin promoter by photo-oxidative mechanisms, it is evident that photo-oxidative-related events are a key factor in the expression of fibrillin/CDSP34 genes in plant leaves. These events are likely to link the up-regulation of these genes to environmental stresses that are known to provoke increased AOS formation in the chloroplast (see Results). Interestingly, such events have recently been shown to be involved in the up-regulation of another gene encoding a chloroplastic drought-induced protein related to thioredoxins (Broin et al., 2000).
Since fibrillin/CDSP34 genes are nuclear encoded, their expression appears to be mediated via an unknown signalling pathway, which may originate from increased AOS levels in the chloroplast. H2O2 appears to be a potential candidate for participating in this signalling pathway due to its ability to diffuse across membranes. Recently, H2O2 has been found to be a key determinant in the systemic signalling in Arabidopsis in response to excess light energy (Karpinski et al., 1999) and under oxidative stress, high concentrations of H2O2 can be produced in the chloroplast due to detoxification of O2·- by superoxide dismutase (Smirnoff, 1993). An accumulation of CDSP34 transcript is shown in detached leaves incubated for a few hours in H2O2 at low concentrations (1 and 10 mM), under conditions where neither change in the glutathione oxidation status nor oxidative damage were noticed (data not shown). In contrast, in similar experiments using methyl viologen (1 µM) or GSSG (5 mM), no accumulation of transcript was observed, despite a dramatic increase in the ratio of oxidized to total glutathione with the latter treatment (data not shown). These data indicate that neither O2·-, nor a change in the redox balance are able to directly and rapidly enhance fibrillin/CDSP34 expression, but that H2O2 may act as an essential effector in this regulatory process.
In leaves of transgenic tomato plants carrying the promoter of the pepper fibrillin gene (Kuntz et al., 1998), a strong induction of the promoter activity was observed in response to biotic stress using elicitors released from pathogens such as E. chrysanthemi. A noticeable increase in fibrillin level in leaves of WT tomato infected by this bacteria was also reported. An oxidative burst triggered by host/pathogen interactions, followed in some cases by a more sustainable AOS release, has been well-documented (Doke, 1997; Alvarez and Lamb, 1997). E. chrysanthemi, which was found to be a good inducer of fib, is usually regarded as causing soft-rot disease without host specificity, but a recent report suggests that plants respond to its infection by producing AOS (El Hassouni et al., 1999). Whether H2O2 is also an effector during the biotic stress-related fibrillin gene induction remains to be demonstrated. The fact that this induction upon bacterial infection was detected only in light is unexpected and would suggest that AOS from plastidial origin are involved, as for abiotic stresses. The mechanism linking bacterial infection and plastidial AOS production needs to be further investigated.
A significant induction of the promoter during ageing is also shown here in green organs such as leaves or cotyledons, suggesting that a development-related mechanism also controls the transcription of fibrillin. This would appear to be in agreement with the data reported previously (Monte et al., 1999) which showed up-regulation of the related gene in leaves of S. demissum under conditions favouring tuberization. However, no increase in the CDSP34 abundance in potato plants grown under long days (14 h, conditions inducing tuberization in this species) was observed compared with that of plants grown under short days (8 h, data not shown). The authors would like to emphasize that CDSP34/fibrillin induction during leaf ageing may also reflect a stress situation. It is known that during ageing levels of AOS rise (Leshem, 1988; Strother, 1988), for example, as a consequence of reduced catalase activity (Strother, 1988). In addition, it should be noted that a relationship between development and oxidative mechanisms was also observed for the pepper fibrillin promoter which is induced during fruit development by cellular redox changes (Kuntz et al., 1998). In conclusion, oxidative mechanisms appear to play an important role in the regulation of genes encoding plastid lipid-asociated proteins during environmental stress, but likely also upon organ development.
Different lines of evidence indicate that, in Solanaceae plants, induction of the pepper promoter reflects accumulation of the endogenous fibrillin/CDSP34 protein. For example, fibrillin promoter activity and fibrillin/ CDSP34 protein levels are noticeably increased during leaf ageing in tomato, by wounding or under water deficit conditions in tobacco (Chen et al., 1998). However, post-translational mechanisms participate in the accumulation of fibrillin/CDSP34 proteins in dicots as well. This is the case for ABA, which was suggested to have a post-transcriptional role in CDSP34 protein accumulation (Gillet et al., 1998). Furthermore, it was recently reported that light intensity controls the expression of the pepper fibrillin gene in transgenic tobacco plants via a splicing step (Rey et al., 2000) and in the present study, it was found that the protein accumulation profile in potato under oxidative stress does not strictly parallel mRNA accumulation (Fig. 1). Altogether, these data indicate the existence of multilevel regulations that could enable plants to fine-tune levels of fibrillin/CDSP34 proteins depending on the nature and strength of the applied stress.
Interestingly, a strong increase in the abundance of the related protein was observed in a highly drought-tolerant species, C. plantagineum which can be desiccated up to 1% RWC (Bartels et al., 1990). In this species, despite severe chloroplastic ultrastructural changes during water loss, rehydrated plants were found to rapidly resume full photosynthetic activities (Bartels et al., 1992). Since plant fibrillins have been reported to stabilize hydrophobic carotenoid- or lipid-accumulating structures (Deruère et al., 1994; Ting et al., 1998; Kessler et al., 1999; Vishnevetsy et al., 1999b) it is therefore tempting to propose that fibrillin also participates in the structural stabilization and protection of thylakoids upon water loss in Craterostigma. More generally, it has been suggested that a similar function could also be fulfilled by the protein in other species, helping plants to tolerate, at least temporarily, the effects of oxidative stress (Gillet et al., 1998).
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Acknowledgments |
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The authors wish to thank J Massimino for growing plant material, P Auroy for assistance in Western experiments and Dr M Kazmaier for irradiation experiments. We also thank E Charpentier for technical assistance, Dr J Gaffé for critical reading and Dr G Green for proofreading the ms. The work performed in Grenoble was supported by the European Commission DGXII Biotechnology Programme (contract number BIO-96-2077).
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Notes |
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3 To whom correspondence should be addressed. Fax: +33 476 51 43 36. E-mail: marcel.kuntz{at}ujf\|[hyphen]\|grenoble.fr
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Abbreviations |
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CDSP34, chloroplast drought stress protein of 34 kDa, fib, promoter of the pepper fibrillin gene fused to the GUS reporter gene, GUS, ß-glucuronidase.
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References |
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