JXB Advance Access originally published online on June 18, 2007
Journal of Experimental Botany 2007 58(10):2525-2535; doi:10.1093/jxb/erm122
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RESEARCH PAPER |
Immunomodulation of jasmonate to manipulate the wound response
1Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben, Corrensstrasse 3, D-06466 Gatersleben, Germany
2Leibniz-Institut für Pflanzenbiochemie Halle, Weinberg 3, D-06120 Halle (Saale), Germany
3Lehrstuhl für Pflanzenphysiologie der Ruhruniversität Bochum, Universitätsstraße 150, D-44801 Bochum, Germany
To whom correspondence should be addressed. E-mail: conradu{at}IPK-gatersleben.de
Received 18 January 2007; Accepted 2 May 2007
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Abstract |
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Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.
Key words: Expression analysis, immunomodulation, jasmonate, wound response
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Introduction |
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Plants have to adapt constantly to fluctuations of their biotic and abiotic environments. Jasmonates enable plants to respond to biotic threats such as mechanical injury, herbivore and pathogen attack, or osmotic, salt, or desiccation stress (Wasternack and Hause, 2002). Moreover, some stages in plant development such as pollen formation, seed germination, or root growth are influenced by jasmonates.
Jasmonates are lipid-derived compounds, originating from 
-linolenic acid in chloroplast membranes. In a 13-lipoxygenase (13-LOX)-catalysed reaction, molecular oxygen is inserted into carbon atom 13 of -linolenic acid. The resulting 13-hydroperoxide is the substrate of at least seven different enzymes of the so-called LOX pathway (Feussner and Wasternack, 2002). In the allene oxide synthase (AOS) branch, an unstable allene oxide is formed and converted into the stable cis-(9S,13S)-12-oxophytodienoic acid (OPDA) by allene oxide cyclase (AOC). OPDA, as well as its derivatives, collectively called octadecanoids, carries the enantiomeric structure of the naturally occurring jasmonic acid (JA), and is formed within chloroplasts (Feussner and Wasternack, 2002). How OPDA is released from the chloroplast is still unclear. The subsequent steps in JA biosynthesis, reduction by OPDA reductase 3 (OPR3) and β-oxidation, were shown to occur in peroxisomes (Stintzi and Browse, 2000; Strassner et al., 2002; Castillo et al., 2004; Li et al., 2005; Theodoulou et al., 2005). Free JA is the main fraction among JA compounds and can be metabolized to its methyl ester (JAME) (Seo et al., 2001), its amino acid conjugates (Kramell et al., 1997; Staswick et al., 2002), and to 12-hydroxy-JA (Gidda et al., 2003).
Elucidation of the role of JA by manipulating JA levels has been addressed by mutant analyses and transgenic approaches. In addition to JA signalling mutants (Staswick et al., 1992, 2002; Berger et al., 1996; Xie et al., 1998; Feys et al., 1994), defects in JA biosynthesis and metabolism (McConn et al., 1997; Sanders et al., 2000; Stintzi and Browse, 2000; Park et al., 2002; von Malek et al., 2002) as well as mutants with constitutively elevated JA levels were described (Hilpert et al., 2001; Ellis et al., 2002). Transgenic approaches to alter JA biosynthesis were performed either to increase or to repress expression of AOS and AOC, respectively. Constitutively elevated levels of jasmonates, however, were not found upon overexpression of an AOS in tobacco (Wang et al., 1999; Laudert et al., 2000) or of AOC in tomato (Stenzel et al., 2003a), suggesting that JA biosynthesis is regulated by substrate availability. This is in line with the observation that in leaves of Arabidopsis thaliana, formation of OPDA and JA occurs only upon substrate generation in response to wounding or pathogen attack, although LOX, AOS, and AOC occur abundantly (Stintzi et al., 2001; Stenzel et al., 2003b). By contrast, elevated JA levels were observed in transgenic potato plants overexpressing a plastid-localized AOS (Harms et al., 1995). This increase, however, was not accompanied by constitutive expression of JA-responsive genes, suggesting a sequestration of JA. Obviously, distinct plant species react differently to transgenic alterations in hormone levels (Hedden and Phillips, 2000).
However, transgenic approaches to modify expression of hormone biosynthetic genes do not address the issue of subcellular sites of hormone action. To this end, immunomodulation was successfully applied to analyse the function of abscisic acid (ABA) and gibberellic acid (GA) (Artsaenko et al., 1995; Shimada et al., 1999; Conrad and Manteuffel, 2001).
Here, genes are used which code for a recombinant single-chain variable fragment (scFv) antibody, selected against the naturally occurring enantiomer of JA, (3R,7R)-JA. The scFv was expressed in Nicotiana tabacum plants and targeted into different cellular compartments, i.e. the cytosol and the endoplasmic reticulum (ER). The scFv antibodies were found to be correctly targeted and functionally active. Macroarray analysis and JA measurements in mechanically wounded transgenic plants revealed compartment-specific immunomodulation of jasmonate levels and functions.
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Construction of expression cassettes
Anti-JA and anti-OPDA scFv antibodies were selected from Tomlinson's human synthetic VH+VL scFv phage libraries A+B (Winter et al., 1994). The screening was done against JA–bovine serum albumin (BSA) conjugates. The gene coding for anti-JA scFv was either directly inserted in the NcoI/NotI sites of the pRTRA7/3 vector (Artsaenko, 1996) for targeting in the cell cytosol, or was amplified by PCR as an anti-JA scFv–c-myc fusion and inserted in the BamHI sites of the expression cassette for scFv retention in the ER (Artsaenko et al., 1995).
Plant material and treatments
Transgenic Nicotiana tabacum cv. Samsun NN plants were generated by Agrobacterium-mediated gene transfer (Zambryski et al., 1983). Plants arising from callus were designated as F0, and plants growing from seeds of F0 plants were designated as F1. Wild-type and individual F1 transgenic plants were grown from seeds for 1 month on Murashige–Skoog medium with 100 mg l–1 kanamycin in sterile conditions and another 2 months in soil in the phytochamber with controlled conditions (16 h light; humidity, 70%; temperature, 23 °C; fresh air, 40%).
Wild-type and F1 transgenic plants with the highest scFv expression level, determined by western blot analysis, were used for the wound stress experiments. Detached leaves were wounded by perforating the whole leaf surface across the main veins and were kept on humid filter paper in a closed Petri dish for the times indicated. Leaves cut from the same plant and immediately frozen in liquid nitrogen were taken as unwounded controls.
The seed germination experiment was performed with seeds of wild type, seeds of F1 transgenic plants accumulating anti-JA scFv in the cytosol (or in the ER), and seeds of transgenic tobacco plants expressing scFv against 2-phenyl-oxazol-5one (oxazolone) in the ER (Fiedler and Conrad, 1995). Seeds were incubated on Murashige–Skoog medium containing 50 mg l–1 kanamycin and either 0.2 mM JAME or an equal volume of sterile dH2O.
Purification of scFv from plant extract
One millilitre of rProteinLTM coupled to CNBr-activated sepharose (ACTIgen) was transferred into a small column and washed with A-PBS buffer (100 mM Na2HPO4.12H2O, 100 mM NaH2PO4.H2O, 0.15 M NaCl, pH 7.2). Aliquots (5–10 g) of tobacco leaves were ground in liquid nitrogen. The powdered tissue was resuspended in 3 volumes of PBS (0.03 M Na2HPO4.2H2O, 0.017 M NaH2PO4.H2O, 0.1 M NaCl, pH 7.2) containing 0.1% of Triton X-100. The clear supernatant obtained after centrifugation was applied on the protein L column. The column was washed with A-PBS buffer, and scFv antibodies were eluted with 0.1 M glycine, pH 2.5, into 2 ml tubes each containing 60 µl of 1 M TRIS. The column was washed with A-PBS buffer followed by 22% ethanol in A-PBS buffer. The fractions containing scFv antibodies, detected by western blot, were concentrated in PEG 6000 for 3 h in a dialysing tube.
ELISA with soluble anti-JA scFv purified from plant extracts
A microtitre plate was coated overnight at room temperature with 10 µg ml–1 BSA or with phytohormone–BSA conjugate diluted in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Dilutions of the conjugates were as follows: JA–BSA and OPDA–BSA, 50 µg ml–1; 24-epi-brassinolide–BSA, 1:200; dihydrozeatin riboside–BSA (Erlanger and Beiser, 1964), 1:200; ABA–BSA (Artsaenko, 1996), 1:2000. After blocking unspecific binding sites with 2% BSA in PBS for 2 h, aliquots of the plant-purified anti-jasmonate scFv at a concentration of 1 µg ml–1 in PBS were added and incubated for 1 h at 25 °C. Anti-c-myc antibodies 9E10 (Munroe and Pelham, 1986) diluted 1:100 in 2% BSA in PBS followed by anti-mouse IgG–ALP conjugate (Sigma) diluted 1:2000 in 2% BSA in PBS were used for their detection. The enzymatic reaction was visualized by 1 mg ml–1 p-nitrophenyl phosphate (Sigma). The intensity of yellow colour developed after 1 h was measured at 405 nm with the ELISA reader Dynatech MR 7000.
Immunofluorescence
Small pieces of leaves of 8-week-old wild-type and transgenic tobacco plants were fixed with 4% (w/v) paraformaldehyde and 0.1% (v/v) Triton X-100 in PBS (135 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4). After dehydration in a graded series of ethanol, material was embedded in PEG and cut as described (Hause et al., 1996). Cross-sections (2 µm thick) of leaves were immuno-labelled simultaneously with monoclonal mouse anti-myc antibody 9E10 [diluted 1:10 in PBS containing 1 % (w/v) acetylated BSA and 1 mg ml–1 goat IgG] or with rabbit anti-BiP antibody [diluted 1:500 in PBS containing 1% (w/v) BSA, kindly provided by Jürgen Denecke, University of Leeds, UK]. As secondary antibodies, a goat anti-mouse IgG conjugated with AlexaFluor 488 and a goat anti-rabbit IgG conjugated with AlexaFluor 543 (both Molecular Probes, Eugene, OR, USA) were used at a the dilution of 1:500. After immunolabelling, sections were stained with 0.1 µg ml–1 DAPI (4,6-diamidino-2-phenylindole; Sigma, Taufkirchen, Gemany). Immunocytochemical stainings were analysed with the Axioskop epifluorescence microscope (Carl Zeiss, Jena, Germany) using the proper filter combination. Confocal imaging was performed using a Zeiss LSM510 META using multi-tracking mode. Co-localization analysis was performed using Zeiss LSM-software. All micrographs were processed through the Photoshop 8.0.1 program (Adobe, Seattle, WA, USA).
Western blot analysis
Denaturated protein extracts were obtained from tobacco leaf discs (
1 cm) extracted in SDS–sample buffer (1 ml glycerol, 1.4 ml 0.5 M TRIS–HCl, pH 6.8, 2 ml 10% SDS, 0.5 ml β-mercaptoethanol, 5.1 ml ddH2O, 0.001% bromphenol blue) and boiled for 10 min. The protein content was determined by the Bio-Rad Protein Assay. Twenty micrograms of total soluble protein were separated by SDS–PAGE, followed by electrotransfer to Protran® nitrocellulose transfer membrane (Sambrook et al., 1989). Anti-c-myc primary antibody diluted 1:50 and anti-mouse IgG-HRP (Amersham) secondary antibody diluted 1:2000 were used for the detection. The scFv protein was visualized with the ECL western blotting analysis system (Amersham).
Northern blot analysis
Total RNA was isolated from plant tissue according to Heim et al. (1993). Ten micrograms of the total RNA were separated in 1% agarose–formaldehyde gel, transferred to Hybond N+, a positively charged nylon membrane (Amersham), and hybridized with DNA of a wound stress-specific gene radioactively labelled with [-33P]dCTP using the Megaprime DNA labelling kit (Amersham). Hybridization was carried out in the Roti-Hybri-Quick solution (Roth) overnight at 65 °C. Post-hybridization washing was performed at 65 °C stepwise in 2x SSC, 1x SSC, and twice in 0.2x SSC, always with 0.1% SDS. The membrane was exposed to an imaging plate (BAS-III; Fuji foto film, Japan) and radioactive images were obtained with phosphoimager Storm 860 (Molecular Dynamics, USA).
Macroarray analysis
DNA spotting:
Plasmid DNA containing genes of interest was diluted in 50% TE buffer for final concentrations of 1500 ng, 750 ng, and 375 ng, and spotted with the spotting robot BG600 (BioGrid, GB) on Hybond N+. The positively charged nylon membranes (Amersham) with the three concentrations of DNA were next to each other. ‘Empty’ pBluescript vector and clones containing chicken DNA fragments not known to be present in the plant genome also spotted in the three concentrations were included as negative controls. Filters denaturated in the denaturation solution (1 M NaCl, 0.4 M NaOH) and neutralized twice in the neutralization solution (1 M NaCl, 0.5 M TRIS–HCl, pH 7.5) were briefly dried and DNA fixed on the membranes by UV cross-link.
Synthesis of labelled cDNA:
Dynabeads® mRNA purification kit (Dynal, Norway) was used for the purification of mRNA from the total RNA isolated from tobacco leaf tissue as described elsewhere in Materials and methods. mRNA bound to Dynabeads Oligo (dT)25 was reverse transcribed by use of SuperscriptTM II RNase H– reverse transcriptase (GibcoBRL). The second strand cDNA, labelled with [-33P]dCTP, was synthesized using the Megaprime DNA labelling kit (Amersham). The labelled cDNA was eluted from the Dynabeads and applied on VectaSpin MicroTM centrifuge tube filters with a pore size 0.2 µm and an Anopore membrane (Whatman®, USA) to separate the remaining beads. Labelled cDNA was hybridized with the macroarrray filters as described elsewhere in Materials and methods.
Identification of wound-responsive genes
The quantification of the signal intensity was carried out using Array Vision software (Amersham Pharmacia Biotech, USA). Relative signal intensity was calculated from the quantified signal intensity for normalization of differences of signal intensities on each filter. A logarithmic value of the ratio of each signal intensity on filter A to the corresponding signal intensity on filter B chosen as the reference filter was calculated. The median of the ratios of all spots on filter A was determined and the antilogarithmic value represented the normalization factor of filter A. The relative signal intensity of each spot of filter A was then calculated as the ratio of each signal to the normalization factor of filter A. Only those genes whose relative signal intensity grew linearly with the corresponding increasing concentration of the spotted DNA were taken into account.
Determination of endogenous levels of phytohormones
About 0.5 g of tobacco leaf was used for the analysis. The extraction and measurement were carried out as described in Müller et al. (2002).
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Expression of anti-JA scFv antibodies in the cytosol and in the ER of transgenic tobacco plants
Anti-JA-specific recombinant single-chain Fv antibodies were obtained by screening the Tomlinson's human synthetic VH+VL scFv phagemid library (see Materials and methods) with (3R,7R)-JA coupled to BSA. After selection of phages displaying JA-binding activities, the coding regions of the respective antibodies were cloned behind the cauliflower mosaic virus 35S (CaMV35S) promoter in expression cassettes for plant transformation (Fig. 1). Thus, expression of the construct shown in Fig. 1a should allow cytosolic accumulation of the antibodies. The KDEL signal at the C-terminus of the scFv should enhance accumulation in the cytosol (Schouten et al., 1997). To target the antibodies to the ER, fusion of the legumin B4 signal peptide to the N-terminus and the presence of the ER retention signal KDEL should result in an accumulation of scFv in the ER (Fig. 1b). For both constructs, the c-myc tag peptide was included to allow easy detection and purification of the scFv produced in the transgenic plants.
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Characterization of transgenic tobacco plants expressing anti-JA antibodies compartment-specifically
Transgenic tobacco plants were generated by leaf disc transformation using Agrobacterium tumefaciens harbouring the anti-JA scFv constructs. Kanamycin-resistant F1 plants were screened by western blot analysis to identify transformants with the highest level of scFv expression in the two cellular compartments. Anti-JA recombinant antibodies accumulated in the cytosol or in the ER to a level of 0.05% of total soluble protein (Fig. 2).
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To prove the localization of the anti-JA scFv in the ER, immunocytological analysis was performed. The scFv detected via the c-myc-tag co-localizes with the ER marker BiP (Fig. 3B, C). The immunocytological analysis of leaves of wild-type plants revealed only the autofluorescence of the tobacco chloroplasts (Fig. 3A).
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Antigen-binding activity of the plant-produced anti-JA scFv was determined in crude leaf protein extracts of T1 plants purified via affinity matrix columns. The phytohormones JA, 24-epi-brassinolide, dihydrozeatin riboside, and ABA coupled to the BSA carrier were used to demonstrate the binding specificity of the plant-produced scFvs. First, the specificity of each phytohormone conjugate was verified by ELISAs which showed exclusive binding of the BSA-coupled antigens by their cognate antibodies (data not shown). The binding specificity of the immunopurified antibodies from plants was shown by ELISA (Fig. 4). The anti-JA scFv bound exclusively to BSA-coupled JA, suggesting that the plant-produced antibodies specifically recognize jasmonates.
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The anti-JA scFv transgenic tobacco plants contain less physiologically active JA
To investigate whether the high levels of anti-JA scFv in transgenic plants and its ability to bind JA result in reduced levels of physiologically active JA, inhibition of seed germination in response to JA treatment and expression of JA-responsive genes upon wounding were analysed. Seeds of wild-type and transgenic tobacco plants expressing ER-localized scFv antibodies against 2-phenyl-oxazol-5one (oxazolone), a compound not naturally present in plants, were used as a control. Seeds of transgenic plants accumulating the anti-JA scFv in the cytosol or in the ER, as well as the control seeds, germinated equally well on Murashige–Skoog medium without JAME (Fig. 5). As expected, germination of control seeds was strongly inhibited by treatment with 10 µM JAME. Germination and growth of transgenic plants, however, were significantly less inhibited by JAME treatment. In the case of the transgenic plants with cytosolic expression of anti-JA scFv, germination inhibition was no longer detectable. These results suggest that the accumulated anti-JA antibodies are functional, leading to reduced amounts of physiologically active JA.
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Expression of many wound-responsive genes depends on a transient rise in JA content (Howe, 2005). Therefore, macroarray analyses were performed to analyse whether the expression of anti-JA scFv antibodies in tobacco alters expression of JA-responsive genes. Two hundred and fifty genes, falling into different groups according to the putative biological function of their encoded proteins, were spotted onto nylon membranes and were hybridized with radioactively labelled cDNA derived from detached leaves of wild-type or transgenic plants wounded for 24 h. Figure 6 shows the expression of genes that were reproducibly wound-induced at least 10-fold in the wild type. These include the wound-induced proteinase inhibitor II gene (Nt-PIN2, Z29537.1), PR genes encoding osmotin (PR5, AY737310.1), PR-1b (X17680.1), and SAR8.2 (U64809.1), as well as an extensin (M34371). In addition to being salicylic acid-inducible, PR-1 and PR5 genes are expressed in response to ethylene and JAME treatment in tobacco (Xu et al., 1994). EAS (L04680.1) and HMGCoA reductase (AF004232.1) encoding genes were also induced by wounding in detached tobacco leaves.
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Except for extensin, expression of the anti-JA scFv antibodies in the cytosol and in the ER caused a significant reduction in wound-induced expression of all genes analysed. In the case of extensin no reduction could be detected in leaf tissue expressing the anti-JA scFv antibodies in the ER. These macroarray hybridization data could be validated by northern blot analyses as exemplified for NtPIN2, OSMOTIN, and EXTENSIN (Fig. 7). Both methods revealed reduced expression of NtPIN2 and OSMOTIN upon wounding of transgenic tobacco plants expressing anti-JA scFv antibodies in the cytosol and in the ER.
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To test whether this reduced gene expression can be normalized by JAME treatment, leaves of the transgenic plants were wounded and incubated for 24 h or 48 h with or without 200 µM JAME. The subsequent macroarray and northern blot analyses revealed complete normalization of NtPIN2 expression in plants expressing anti-JA scFv in the cytosol and only about 50% normalization in the case of ER-localized anti-JA scFv (Fig. 8). Only partial normalization was observed in the case of the other defence genes (data not shown).
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Analysis of endogenous levels of jasmonic acid
Endogenous levels of JA were analysed quantitatively by gas chromatography/ion-trap mass spectrometry. Detached leaves of wild-type tobacco and transgenic plants expressing anti-JA scFv were examined before wounding, as well as 2 h and 24 h after mechanical wounding.
Elevation in the endogenous JA level was detectable at 2 h after wounding whereas basal JA levels were reached 24 h after wounding in wild-type as well as in the transgenic plants (Fig. 9). Interestingly, JA levels of transgenic plants expressing anti-JA scFv in the cytosol and in the ER, respectively, exceeded the levels detectable in the wild type up to 40-fold. By contrast, endogenous levels of other acidic phytohormones such as OPDA, salicylic acid, ABA, and indole-3-acetic acid did not differ between the wild-type and transgenic plants (data not shown). These results indicate that elevated JA levels observed in the transgenic plants are not a consequence of changes in endogenous levels of other acidic phytohormones, but are rather due to expression of the anti-JA antibodies.
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Discussion |
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Immunomodulation, originally developed for human cells, is a molecular technique by which cellular metabolism, signal transduction pathways, or pathogen activities can be interfered with by the expression of a gene encoding a recombinant antibody directed against target molecules such as proteins, hormones, or secondary metabolites. Several models have been proposed suggesting how immunomodulation could function in plants (De Jaeger et al., 2000, Conrad and Manteuffel, 2001). In the case of small molecules, such as phytohormones or secondary metabolites, direct binding of the active compound by the antibody would reduce the functional amount of the compound, thus interfering with its function. Importantly, the affinity of the plant-expressed antibody must be high enough to compete with putative hormone receptors or binding proteins. Since binding of the physiologically active form of any phytohormone should occur at the site of action, alterations in phytohormone responses in an immunomodulated plant will give indications regarding phytohormone activity. By contrast, analyses of hormone action by transgenic manipulation of phytohormone synthesis or transport may lead to side-effects which are difficult to control. Alternative methods have been successfully applied in various studies. Among them is down-regulation of transcript accumulation by double-stranded RNA interference or the introduction of a cDNA coding for a novel enzyme catabolizing the compound of interest (Hedden and Phillips, 2000). However, redundancies and differential substrate specificities of enzymes or novel degradation products may lead to unwanted effects. Removal of GA intermediates by overexpression of a catabolizing enzyme, for example, yielded only a weak phenotype because of a partial compensation due to the up-regulation of GA biosynthetic enzymes (Xu et al., 1999).
In immunomodulation, hormone levels can even be modulated in a compartment-specific manner. Recombinant antibodies targeted to specific cell compartments by using different signal or transit peptides (Conrad and Manteuffel, 2001; Jobling et al., 2003) may create artificial sinks for functional molecules, such as phytohormones, and could thus cause deficiency in other compartments. This approach has been successfully applied to modulate the action of ABA in seeds of tobacco and in leaves of tobacco and potato (Artsaenko et al., 1995; Phillips et al., 1997; Strauss et al., 2001). In all of these cases, the anti-ABA-scFv was targeted to the ER. Analyses of ABA and scFv distribution in transgenic potato leaves by non-aqueous fractionation supported the idea of an artificial ‘ABA sink’ in the ER (Strauss et al., 2001). Moreover, in some cases of ABA deficiency following immunomodulation, stronger phenotypes were obtained than with mutants of ABA synthesis. This might be caused by the fact that ABA mutants are leaky and, in the case of embryo development, maternal effects could mask ABA deficiency (Senger et al., 2001). Therefore, the creation of an artificial sink in the ER, as well as the direct binding of target molecules in the cytosol, were chosen to modulate the amount of functionally active JA for analyses of its action.
The present study was designed to find functional consequences of compartment-specific expression of anti-JA scFv. As a prerequisite, recombinant anti-JA scFv was shown to bind JA. Tobacco plants expressing anti-JA scFv in both the cytosol and the ER of transgenic plants were generated, and the correct targeting of the antibodies was shown immunocytochemically (Fig. 3). Two JA-dependent responses were recorded to assess the amount of physiologically active JA: (i) insensitivity of germinating transgenic seedlings towards JAME; and (ii) loss of wound-induced gene expression. The latter phenotype could be by-passed by exogenous application of an excess amount of JAME (Fig. 8A). Plant-expressed scFv were able to bind JA, suggesting that the phenotypes were caused by binding of JA to the antibodies expressed in the cytosol or the ER. That JAME can be used to complement the JA-deficient phenotypes is based on the following observations: (i) intracellularly there is an equilibrium of JA and JAME with preferential occurrence of JA; (ii) similar to JA, it is possible to bind JAME with recombinant anti-JA scFv antibodies (data not shown); (iii) dose–response curves and time-course analyses for JA- and JAME-induced expression of JA-activated genes revealed a common expression pattern with weakly preferential inducibility by JAME (Miersch et al., 1999; Kramell et al., 2000; Taki et al., 2005).
Similar to anti-ABA-scFv, anti-JA scFv accumulating in the ER may trap JA in this compartment and may form an artificial sink. On the other hand, cytosol-localized anti-JA scFv may bind JA in the cytosol and thus may affect subsequent signalling or binding to a putative receptor. This would eventually lead to a decrease in jasmonate-induced gene expression or other JA-dependent processes in these transgenic plants. The JA-deficient phenotype observed, together with the fact that wild type-like PIN2 expression could be achieved upon JAME treatment, suggests that the wound response requires cytosolic JA. One should stress here that the hypothetically antibody-bound JA and free JA are determined together due to the extraction procedure.
In wild-type plants, JA levels increase rapidly after wounding. This leads to the activation of early wound-responsive genes such as those encoding JA biosynthetic enzymes. Consequently, JA biosynthesis increases by positive feedback regulation as shown by transgenic approaches in Arabidopsis, tobacco, and tomato (Laudert et al., 2000; Stenzel et al., 2003a). Late wound-induced genes such as those encoding proteinase inhibitors are also activated due to a rise in JA levels, leading to the accumulation of PIN2 transcript 24 h after wounding (Fig. 8E). In transgenic plants expressing anti-JA scFv, JA is efficiently bound and removed from the pool of physiologically active JA, which becomes apparent by the low levels of PIN2 transcripts detectable 24 h after wounding (Fig. 8E).
The observation that PIN2 transcript levels are less reduced in transgenic plants expressing anti-JA scFv in the ER (Fig. 8E) indicates that the creation of a sink in the ER is not as effective in reducing the amounts of physiologically active JA as by direct binding of JA by antibodies in the cytosol. Possibly, JA diffuses from the cytosol into the ER where it is trapped by the antibodies. Assuming that this diffusion reaches an equilibrium, residual amounts of JA in the cytosol might still be able to activate gene expression.
The specificity of jasmonate function in the cytosol is supported by another observation. The intracellular accumulation of functional anti-JA antibodies had no impact on the endogenous levels of salicylic acid, ABA, or indole-3-acetic acid (data not shown). Thus, the measured abundance of wound-response gene transcripts is not due to changes in the levels of these compounds.
Interestingly, the low amounts of physiologically active JA are apparently sensed by the anti-JA scFv-expressing plant which reacts with increased biosynthesis of JA. Ultimately, JA accumulates to levels which are 40-fold higher than those detected in wounded wild-type plants (Fig. 9). This drastic increase in JA levels after wounding of scFv-expressing plants indicates the existence of a tight control of JA levels. By contrast to RNA interference of JA biosynthetic enzymes, which decreases the capacity to synthesize JA and subsequently leads to decreased synthesis of OPDA and JA (Stenzel et al., 2003a), immunomodulation removes JA, while fully retaining the plant's capability to synthesize JA. Thus, JA immunomodulation reveals a novel aspect of the plant's ability to sense JA levels. On the one hand, if sufficiently high JA levels are not achieved, the plant reacts with increased synthesis. On the other hand, in addition to positively affecting its own synthesis, JA is also required to limit its own synthesis during the wound response.
This impact of JA immunomodulation on the JA biosynthetic pathway differs from that of ABA. Studies with transgenic plants accumulating anti-ABA scFv in the ER showed a dramatic increase of ABA in untreated transgenic leaves and seeds in comparison with wild-type and control plants (Phillips et al., 1997; Wigger et al., 2002). By contrast, no changes in JA and OPDA content could be measured in non-treated anti-JA scFv-expressing plants. Two hours after wound stress, however, a dramatic transient increase in JA was observed in plants accumulating anti-JA antibodies in the cytosol and in comparison with stressed wild-type plants. A transient increase in OPDA 2 h after wounding was also detected, but two orders of magnitude lower than the JA increase (data not shown).
Possibly, in unwounded plants, increased JA synthesis cannot occur due to a lack of substrate. This limited substrate availability for JA biosynthesis had already been suggested, based on attempts to increase JA levels by overexpressing biosynthetic enzymes. Transgenic tomato plants constitutively overexpressing allene oxide cyclase exhibit elevated JA levels only upon wounding, indicating that substrate availability is regulating JA biosynthesis (Stenzel et al., 2003a, b). Most attempts constitutively to overexpress genes coding for JA biosynthetic enzymes failed to show constitutive elevation of JA but showed a burst of JA upon wounding (Wang et al., 1999; Laudert et al., 2000; Park et al., 2002), in accordance with the situation in the anti-JA scFv plants.
Both strategies, the direct binding of the target molecules in the cytosol and the creation of an artificial sink in the ER, were successful and led to the following new aspects. (i) Expression of anti-JA scFv antibodies in tobacco impedes the jasmonate-dependent wound response and prohibits inhibition of germination by exogenous JAME. (ii) Anti-JA scFv antibodies can be targeted successfully to different cellular compartments, where they hamper jasmonate responsiveness to different extents. Cytosolic JA seems to be preferentially active as shown upon wounding. (iii) The present results suggest that the stress-induced JA level is sustained by a cross-talk between the initial level and JA biosynthesis.
The tobacco lines expressing anti-JA scFv antibodies compartment-specifically are a versatile tool in the future study of plant–pathogen interactions and systemic responses by grafting experiments.
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Acknowledgements |
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The authors would like to thank Dierk Scheel and Elmar W. Weiler for support, Jürgen Denecke for the anti-BiP antibodies, and Mrs Isolde Tillack and Mrs Ursula Schumann for technical help. Financial support was given by the Deutsche Forschungsgemeinschaft SPP 322 1067 to UC and SR.
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* Present address: Department of Developmental Genetics/Section Plant Physiology, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
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