JXB Advance Access originally published online on January 8, 2007
Journal of Experimental Botany 2007 58(3):555-568; doi:10.1093/jxb/erl230
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RESEARCH PAPER |
Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts
Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Universidad Autónoma de Madrid, Carretera de Colmenar Viejo km 15,500. 28049 Madrid, Spain
* Present address and to whom correspondence should be sent. Institute of Agrobiotechnology, Centre for Research and Technology, 6th Km Charilaou-Thermi Rd., 570 01 Thermi, Thessaloniki, Greece. E-mail: mfarmaki{at}certh.gr
Received 17 July 2006; Revised 26 September 2006 Accepted 13 October 2006
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Abstract |
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The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.
Key words: Allene oxide cyclase, allene oxide synthase, hydroperoxide lyase, lipoxygenase, oxylipins, stroma, thylakoid
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Introduction |
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Mechanical wounding in plants produced either by abiotic factors or by herbivory, results in the induction of defence genes, many of which have been identified in the model species Arabidopsis thaliana (Titarenko et al., 1997; Reymond and Farmer, 1998), and other plants such as tomato and potato (Hildmann et al., 1992; Bergey et al., 1996). The activation of wound responsive genes is triggered by a complex signalling network in which the plant hormone jasmonic acid (JA) plays a pivotal role (León et al., 2001; Schilmiller and Howe, 2005).
JA and other compounds, such as aldehydes with antimicrobial and pesticidal activities, are products of the oxylipin pathway of fatty acid metabolism (Feussner and Wasternack, 2002). The first step in the lipoxygenase branch of oxylipin biosynthesis is the introduction of molecular oxygen to either C-9 or C-13 positions of linoleic and linolenic acids catalysed by lipoxygenases (LOX). This is a stereo-specific reaction for which 9- and 13-LOX activities, depending on the position where oxygen is introduced, have been described in different plant species. In potato, 9-LOX is the predominant activity in healthy leaves (Hamberg, 2002). As no 9-hydroperoxide lyase (HPL) activity is present in potato leaves (Vancanneyt et al., 2001), the 9-hydroperoxide products of 9-LOX may be used by 9-divinyl ether synthase to give rise to divinyl ethers with antifungal activities (Weber et al., 1999).
Two distinct 13-LOX genes (LOX H1 and LOX H3) are induced in potato leaves upon wounding (Royo et al., 1996). In the close relative tomato, the expression of the LOX H3 homologue (TomLOXD) is also induced in wounded leaves. The LOX H1 homologue (TomLOXC), however, is not wound-inducible but constitutively expressed in the fruits (Heitz et al., 1997). The 13-hydroperoxy fatty acid products of 13-LOX may subsequently be used by either of two divergent enzymatic activities. On the one hand, allene oxide synthase (AOS) dehydrates 13-hydroperoxy-linolenic acid to produce an unstable allene oxide that is converted by allene oxide cyclase (AOC) to the JA precursor 12-oxo-phytodienoic acid (OPDA). On the other hand, a 13-HPL may cleave 13-hydroperoxides diverting them from JA synthesis to yield 12-oxododecenoic acid and either hexanal or 3-hexenal (C6 aldehydes), depending on whether the 13-hydroperoxide is derived from linoleic or linolenic acids, respectively.
Antisense inhibition of LOX H3 in transgenic potato plants results in a severely reduced wound inducibility of proteinase inhibitors and other JA-responsive genes (Royo et al., 1999). LOX H3 was thus suggested to participate in JA-dependent wound activation, although no differences in jasmonate levels between wild-type and LOX H3-depleted plants could be detected. By contrast, transgenic potato plants in which LOX H1 expression was co-suppressed, had highly reduced levels of C6 aldehydes, but nearly wild-type induction of JA-dependent genes upon wounding (León et al., 2002). Likewise, suppression of TomLOXC expression in tomato led to largely reduced C6 aldehyde contents in both leaves and fruits (Chen et al., 2004). In A. thaliana, co-suppression of a 13-LOX (LOX2) results in a severe reduction of JA levels upon wounding, and the concomitant reduction of wound-inducible gene expression (Bell et al., 1995). However, the C6 aldehyde content of the co-suppressed plants has not been reported. Moreover, a putative role that other 13-LOX isoforms present in A. thaliana may play on C6 aldehyde formation has not been reported.
Whilst LOX H3-depleted potato plants have wild-type levels of C6 aldehydes, LOX H1-depleted plants exhibit nearly wild-type jasmonate levels (León et al., 2002). A metabolic compartmentalization of oxylipin synthesis may thus occur by which 13-hydroperoxides produced by LOX H1 are specific substrates for 13-HPL while AOS activity is restricted to the use of 13-hydroperoxides produced by LOX H3. As a first step to validate the hypothesis of a metabolic compartmentalization in oxylipin biosynthesis, the subcellular localization of the enzymes involved has been investigated.
13-LOXs have previously been reported to localize to chloroplasts of photosynthesizing tissues (Feussner and Wasternack, 2002) and similar observations have been made in the cases of HPL, AOS, and AOC (Maucher et al., 2000; Ziegler et al., 2000; Froehlich et al., 2001). A. thaliana LOX2 also resides in the chloroplast (Bell et al., 1995). In fact, all evidence gathered to date suggests that the 13-LOX pathway occurs in the chloroplast (Liavonchanka and Feussner, 2006) up to formation of the AOC product OPDA, which is transported to peroxisomes (Theodolou et al., 2005) where it is reduced by the peroxisomal reductase OPR3 and subject to ß-oxidation to yield JA (Schaller et al., 2005). On the other hand, C6 aldehyde synthesis is thought to be confined to chloroplasts (Schaller et al., 2005).
The involvement of different subcellular compartments in the synthesis of JA and C6 aldehydes suggests that the LOX pathway has to be exquisitely fine-tuned to proceed efficiently. The location of the enzymes within the organelles may indeed determine when and how precursors are fed into the diverging AOS and HPL branches of the pathway, thus giving rise to products with distinct biological properties. For more than a decade, however, conflicting evidence has accumulated regarding the precise localization of enzymes of the LOX pathway within chloroplasts. In contrast to previous studies showing HPL and LOX bound to thylakoids (Bowsher et al., 1992; Hatanaka, 1993), Blée and Joyard (1996) localized LOX, AOS, and HPL activities to chloroplast envelope fractions. In vitro translated AOS and HPL from tomato were shown to be targeted to the inner and outer chloroplast envelopes, respectively (Froehlich et al., 2001). Consistent with this result, a proteomic study of the A. thaliana chloroplast envelope suggested the localization of AOS (At5g42650) and HPL (At4g15440) to the inner and outer envelope respectively (Froehlich et al., 2003). By contrast, only AOS was detected in the envelope fraction in a previous proteomic study (Ferro et al., 2003). However, recent work (Peltier et al., 2004) using a new fractionation method to study the thylakoid proteome of A. thaliana, has identified AOS (At5g42650) in the thylakoid membrane.
The reasons for the conflicting results obtained in the past may, in part, relate to the use of different plant species in these studies, and the fact that most of the enzymes of the LOX pathway are encoded in gene families (Liavonchanka and Feussner, 2006). Indeed, a given isoform whose function and location is identified in one species may not be a true orthologue of any enzyme identified in a different species. These difficulties in extrapolating data obtained from different species have hindered our knowledge on how this synthetic pathway may operate in vivo.
In this work, evidence obtained from a single plant species for a differential distribution of the LOX pathway enzymes within chloroplasts is presented. In addition, the results obtained suggest a possible mechanism to separate the different synthetic branches physically.
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Materials and methods |
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Plant material
The conditions for cultivation of transgenic and non-transformed potato plants (Solanum tuberosum cv. Desiree) and for wound response experiments have been described previously (Royo et al., 1999). A transgenic potato line expressing the GFP-tagged AOC described below, under the control of the cauliflower mosaic virus 35S promoter and the octopine synthase 3' end was generated. A more thorough description of this line will be published elsewhere.
The A. thaliana cell line T87 was grown in liquid Murashige and Skoog medium (Sigma) supplemented with 3% sucrose, and 1 mg l–1 kinetin and 1 mg l–1 1-naphthalene acetic acid. Flasks were shaken at 100 rpm, in a growth chamber at 22 °C, and with a photoperiod of 16/8 h light/darkness. Every week, one-tenth of the culture was transferred to fresh medium.
Cloning of potato AOS-1, AOS-2 and AOC cDNAs
The cDNAs for potato LOX H1, LOX H3, and HPL have been described already (Royo et al., 1996; Vancanneyt et al., 2001). For AOS-2 cDNA cloning, reverse transcription reaction was performed for 1 h at 37 °C with AMV reverse transcriptase (Gibco-BRL) on total RNA extracted from 4 h wounded potato leaf tissue (10 µg), using AOS-6 primer (5'-GCCCGATCAAAAATCTTCGG-3') designed as the best consensus match for flax (Song et al., 1993), rubber (Pan et al., 1995), and A. thaliana (Laudert et al., 1996) AOS sequences. Upon PCR amplification with primer AOS-3 (5'-AGTCAACATGCCACCGGG-3') and AOS-6, a band of 940 bp was obtained. Sequence analysis revealed high similarity to other AOS cDNAs. The 5' and 3' ends of the cDNA were obtained with the Marathon cDNA amplification kit (Clontech). By using AOS-7 (5'-GAAAGAACACGGTAACCACCGGTGAG-3') and a nested primer AOS-9 (5'-CCACCGGTGAGTTCAGTCGA-3'), a band of 432 bp was obtained that was confirmed to be the 5' end of potato AOS-2 by sequence analysis. Similarly, a 900 bp fragment of the 3' end was obtained using AOS-8 (5'-CCTTCGGCGGGATGAAGATTTTCTTCCC-3') and a nested primer AOS-10 (5'-ATGCTGAAATCGATAGCGAAAGCAGG-3'). The full-length cDNA (1835 bp, GenBank accession number AY135640) was assembled taking advantage of restriction sites present in the overlapping regions of the 5' end and 3' end fragments, SalI and ClaI, respectively, and the central AOS-2 fragment.
For AOS-1 and AOC cDNA cloning, first strand cDNA was obtained with primers derived from the published sequences of potato AOS-1 (accession number AJ457080) and tomato AOC (Ziegler et al., 2000). In the case of AOS-1, forward primer 5'-ATGGCATCAACTTCTCTTTCT-3; and reverse primer 5'-TCAAAAACTGGCTCTTCTCAGAGAAGTTAA-3' were used for PCR amplification. In the case of AOC, 5'-CCATGGCCACTGTTTCCTCAGCCTCTGCT-3' and 5'-CACCATGGTAGTGTAATTTTTCAGTGCGGC-3' were used as forward and reverse primers, respectively, for PCR amplification. Sequencing was used to confirm the identity of the amplified fragments (GenBank accession number AY135641).
Antibody production and protein analyses
Antibodies raised against LOX H1 and against synthetic peptides derived from LOX H3 have been described previously (Royo et al., 1999).
The cDNA fragments of LOX H3, AOS-2, AOC and HPL were amplified by PCR using specific primers that introduced a NcoI site at 5' and 3' ends and eliminated their stop codons, and were cloned in-frame in the NcoI site of the corresponding pRSET vector (Invitrogen). After transformation in Escherichia coli BL21, proteins were overexpressed following addition of IPTG (Calbiochem) to the bacterial culture. Histidine-tagged proteins were purified using a nickel resin (Qiagen, Germany), and used to immunize New Zealand rabbits and Wistar rats. Serum from third bleeding was used at appropriate dilutions for western blotting (routinely 1:2500) and electron microscopy histochemical assays (routinely 1:50).
Protein extraction and western blotting were as described (Dammann et al., 1997).
Chloroplast isolation and fractionation
Chloroplasts were isolated in Percoll (Amersham-Pharmacia Biotech) gradients as described by León et al. (2002). Following hypotonic lysis of entire chloroplasts in HEPES–KOH buffer pH 8.0, membranes and soluble components were separated on a step sucrose gradient as described (Froehlich et al., 2001).
Hydroperoxide lyase activity assay
Protein extracts were prepared from chloroplast stroma, envelope, and thylakoid fractions. The protein concentration in the envelope fraction was routinely a thousand times lower than in the stroma and thylakoid ones. HPL activity in each of these fractions was determined by headspace-gas chromatography analysis of the formed aldehydes as described (Vancanneyt et al., 2001) using 50 µg protein (0.5 µg in the case of envelope fractions).
Transient expression of GFP-tagged proteins
LOX H1, LOX H3, HPL, AOS-1, AOS-2, and AOC cDNAs were isolated as NcoI fragments, and cloned in the NcoI site of pMON30063 (Pang et al., 1996) to get the C termini of the corresponding coding regions fused in-frame to the amino terminal end of GFP.
A. thaliana Tic40 (At5g16620), OEP7 (At3g52420), and CAB 180 (At1g29930) cDNAs were amplified with primers designed to match the published sequences, and simultaneously introducing a NcoI site for cloning purposes. They were cloned in the pMON30063 vector as described above.
GFP-tagged proteins were expressed in A. thaliana T87 cells using a biolistic (Bio-Rad) approach for transient transformation (Klein et al., 1987). Transformed cells were observed in a fluorescence microscope (Leica DMR). Single optical sections were obtained using a confocal laser microscope (Zeiss Axiovert 200, Radiance 2000, BIO-RAD) at a 1024x1024 resolution at distances of 0.2 µm. GFP fluorescence was observed with an Argon 488 laser and a band pass emission filter HQ515/30. A dichroic mirror 560 DCLPxR was used. The red autofluorescence of chloroplasts was observed with a laser Helium-Neon 543 nm and the emission filter E570LP. In this case, the dichroic mirror 650 DCLPxR was used. The data were processed using Adobe (Mountain View, CA) Photoshop software and presented in pseudocolour format.
Electron microscopy
Potato leaf tissue was cut in 2 mm2 pieces and vacuum infiltrated with 4% paraformaldehyde, 0.1% glutaraldehyde, and 1% sucrose in 0.1 M phosphate buffer, pH 7.2. Following overnight fixation at 4 °C, leaf fragments were cryo-fixed by plunge freezing in liquid nitrogen-precooled liquid propane in a Leica KF 80, (Austria). Samples were transferred to an automatic freeze substitution unit (Leica EM, AFS, Austria) and freeze substituted for 50 h with methanol containing 0.5% uranyl acetate. Following substitution with pure methanol, the temperature was gradually increased up to –35 °C. Samples were infiltrated with an increasing concentration of Lowicryl K4M and polymerized under UV light for 48 h at –35 °C and 24 h at 22 °C. Ultrathin sections were cut using a microtome (Reichert-Jung, Leica, Austria) and passed onto gold grids. After blocking with 4% bovine serum albumin, proteins were detected with the primary antibodies described above, and 10 nm gold conjugated goat antirabbit antibodies (B.B. International, UK) were used to visualize primary antibodies. Staining was performed by incubation with saturated uranyl acetate and, subsequently, with lead citrate. Samples were analysed on a Jeol 1200 electron microscope.
Entire chloroplasts isolated on Percoll gradients were also fixed as described above (except that no vacuum was applied), cryoprotected by infusion with 2.3 M sucrose, mounted on metal specimen stubs, and frozen in liquid nitrogen. Ultrathin cryosections were prepared using a FC4E cryochamber (Reichert-Jung, Leica, Austria) and immunolabelled as described above.
For double labelling experiments of chloroplast ultrathin sections, rabbit and rat antibodies were mixed. Gold-conjugated anti-rat (5 nm particle size) and anti-rabbit (10 nm particle size) secondary antibodies were also mixed. Staining was performed on ice using a 9:1 mixture of 1% methylcellulose and uranyl acetate.
Labelling density in the different cell organelles, chloroplast thylakoid membrane and stroma, was calculated using a grid point counting method (Lucocq, 1992).
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Patterns of LOXH1, LOXH3, HPL, AOS, and AOC protein accumulation upon wounding
The accumulation of LOX H1 and LOX H3, AOS, AOC, and HPL in potato leaves in response to wounding was studied by western blotting. Prior to mechanical damage, the five enzymes analysed are present in the potato leaves albeit at different levels (Fig. 1, 0 h). While LOX H1 and HPL are easily detected in extracts from non-damaged leaves, LOX H3, AOS, and AOC only accumulate to a low basal level.
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It was previously shown that upon wounding the amount of both LOX H1 and LOX H3 transcripts increased in the leaves following distinct time-courses (Royo et al., 1996). However, LOX H1 protein levels remain constant (León et al., 2002), as it is also the case for HPL (Fig. 1). By contrast, LOX H3, AOS, and AOC protein levels increase in response to wounding (Fig. 1, lanes 2, 6, and 24 h).
Western blotting experiments with the corresponding recombinant proteins confirmed the specificity of the antibodies used for the proteins against which they were raised (results not shown). The specificity of the antibodies was further assessed taking advantage of the available transgenic potato lines engineered for largely reduced levels of the corresponding protein (Royo et al., 1999; Vancanneyt et al., 2001; León et al., 2002; and unpublished results). Western blotting experiments with these transgenic lines showed that the antibodies did not recognize additional epitopes in plant extracts (not shown).
Moreover, results from western blotting with a polyclonal antibody raised against the entire LOX H3 protein (that cross-reacts with LOX H1) suggest that LOX H1 is much more abundant than LOX H3 and/or this latter protein is rather unstable during the extraction procedures (results not shown).
For AOS, the corresponding polyclonal antibody recognizes in western blots two proteins of similar size which may either represent a modification of the enzyme following removal of the signal peptide or recognition of the two AOS isoforms (AOS-1 and AOS-2, accession numbers AY135640 [GenBank] and AJ457080 [GenBank] ) known to exist in potato leaves. Both bands can only be separated after prolonged gel electrophoresis (see Fig. 4). Analyses of plants in which AOS-1 has been specifically silenced indicate that the two bands represent the two AOS isoforms and not an unmodified and modified form of the same protein (P Jimenez et al., unpublished data). Taken together, these results suggest that the antibody used recognizes to similar extent both AOS-1 and AOS-2 isoforms, and that AOS-2 is the most abundant of both. A detailed characterization of the AOS co-suppressed plants will be published elsewhere.
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In all cases, the corresponding preimmune sera did not show any significant reactivity against plant extracts under the conditions used (not shown).
Subcellular localization of oxylipin enzymes by transient expression of GFP-tagged proteins
Potato LOX H1, LOX H3, AOS, and AOC, all contain transit peptide sequences for chloroplast import. In the case of HPL, however, a chloroplast signal peptide cannot be unequivocally identified (not shown).
In order to assess the chloroplastic localization of these enzymes, their cDNAs were tagged at their carboxy termini with green fluorescence protein (GFP) and the localization of the fusion proteins was analysed by transient expression in A. thaliana cell suspension cultures. All gene products localized to discrete structures, which co-localized with the red autofluorescence of chlorophyll, a hallmark of chloroplasts (Fig. 2). Fluorescent spots were never observed in other subcellular locations (i.e. cytoplasm). However, clear differences between the patterns of protein accumulation of the five enzymes could be observed.
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LOX H1- and LOX H3-GFP fusion proteins gave a diffuse fluorescence within chloroplasts. Green dots that localized with the red fluorescence of chloroplasts were also observed (Fig. 3).
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Fluorescence from AOS-1–, AOS-2–, and HPL–GFP fusion proteins was restricted to a few dots within the chloroplast. AOS-2 and HPL gave very similar localization patterns, with strong speckled fluorescence that fully co-localized with the red fluorescence of chlorophyll (Fig. 3). Although AOS-1–GFP fluorescence also gave a dotted pattern, it was clearly of a weaker intensity than AOS-2–GFP, suggesting a higher instability of the AOS-1–GFP fusion protein. In contrast to AOS-2–GFP, some spots of AOS-1–GFP were not intimately associated to the red fluorescence of chlorophyll (Fig. 3). In addition AOS-1 fluorescence was distributed in smaller and more numerous dots compared to HPL, AOS-2 and photosystem II. These putative differences in distribution between AOS-1 and AOS-2 will require further investigation.
AOC–GFP fluorescence was evenly distributed throughout the entire chloroplast. A similar fluorescence pattern has been described for the endogenous tomato AOC (Ziegler et al., 2000). A view through the serial sections of the confocal microscopy shows that the over-expressed AOC localizes to the stroma of the chloroplast overlapping to some extent with the auto-fluorescence of the thylakoid (Fig. 3).
Well-established thylakoid and chloroplast envelope marker proteins fused to GFP were used to assess the validity of this transient transformation assay for suborganellar localization of proteins. Tic40 (At5g16620) and OEP7 (At3g52420) were used as inner and outer envelope markers, respectively (Lee et al., 2001; Heins et al., 2002), resulting in a characteristic ring-like fluorescence (Fig. 3). This pattern has also been observed following stable transformation of the envelope-specific protein Tic55-GFP fusion in transgenic plants (Yamasato et al., 2005). The photosystem II protein CAB180 (At1g29930) was used as a thylakoid marker. It co-localized with the red auto-fluorescence of the thylakoid membrane and appeared as a big dotted structure. Thus, the transient transformation assay of GFP fusions faithfully reproduces the localization of the native cognate proteins.
An ultrastructural analysis of the A. thaliana cells used in this study showed that each cell contained chloroplasts of 1–2 µm average size and each chloroplast contained 1–4 grana maximum. Their size and structure shows remarkable differences to thylakoids of potato leaves (not shown).
Suborganellar localization of the lipoxygenase pathway enzymes using chloroplast fractionation
A more detailed localization of the enzymes involved in the lipoxygenase pathway was obtained by fractionation of potato chloroplasts and subsequent analysis by western blotting.
Isolated intact chloroplasts were lysed and fractionated through sucrose gradients into stroma, envelope membrane, and thylakoid fractions. Western blotting showed that both HPL and AOS are associated with the thylakoid-enriched interphase of the gradient (T in Fig. 4). In this case, upon hybridization with the AOS antibody, bands for both AOS-1 (upper band) and AOS-2 (lower band) can be distinguished in the thylakoid fraction. In some experiments, HPL and AOS antibodies also detected the presence of cross-reacting proteins in the envelope fraction, although much less abundant than in thylakoids (see lane E in Fig. 4). These bands may represent non-processed forms still associated with the chloroplast envelope, although this point has not been investigated in detail.
LOX H1 and AOC proteins could also be detected in the thylakoid fraction. However, in contrast to AOS and HPL, a large part of LOX H1 and AOC was found in the soluble, stromal fraction of the chloroplasts (S in Fig. 4). The suborganellar localization of AOC was confirmed taking advantage of a transgenic potato line expressing a GFP-tagged AOC (P Jimenez et al., unpublished results). With an antibody that recognizes GFP, a band of the size expected for the AOC-GFP fusion protein is detected in both stromal and thylakoid fractions. This band is also observed with the antibody raised against AOC (not shown). This result clearly shows that one and the same protein localizes to both stromal and thylakoid fractions of the chloroplast, and suggests that partition of AOC (and perhaps LOX H1) between stroma and thylakoid may be subject to a dynamic process in response to hitherto uncharacterized factors.
The detection of LOX H3 was not possible, due perhaps to a rapid degradation of the protein during prolonged chloroplast isolation and extraction procedures.
The identity and purity of the fractions were determined using DD1 (E Vancanneyt and Sánchez-Serrano, unpublished results) as a marker of the stroma, TOC75 as an outer membrane envelope marker (Tranel et al., 1995), TIC110 and TIC40 as inner membrane envelope markers (Heins et al., 2002), and the light-harvesting complex II protein as thylakoid marker (Viro and Kloppstech, 1980), and further verified by electron microscopy of resin embedded sections (not shown). These results indicate that AOS and HPL are membrane-bound proteins whilst LOX H1 and AOC are present in both membrane and soluble fractions. Moreover, these results show that chloroplast envelopes only contain residual amounts of the LOX pathway proteins.
However, it has been reported that the enzymatic activities of the lipoxygenase pathway largely reside in the chloroplast envelope membrane (Blée and Joyard, 1996). This localization could imply that the lipoxygenase pathway proteins detected in the thylakoid fractions in the present work were enzymatically inactive. HPL activity in the stromal, thylakoid, and envelope fractions was therefore determined. Whilst thylakoid fractions exhibited high HPL activity (617.4±14.8 nmol min–1 mg–1 protein), consistent with the western blot analysis, this enzymatic activity was below detection levels in both stromal and envelope fractions (at least 10-fold less activity). This result suggests that a significant part of the lipoxygenase pathway activity may indeed be associated to thylakoids.
Association of the lipoxygenase pathway to thylakoid membranes
The thylakoid membrane was thus shown to contain many of the enzymes of the lipoxygenase pathway of oxylipin metabolism. The strength of the interaction of the different enzymes with thylakoids was further analysed by the incubation of membrane fractions (León et al., 2002) with different concentrations of either NaCl or Triton X-100 followed by western blotting (Fig. 5). Association of AOS and HPL to thylakoid membranes was resistant to high-salt treatment. By contrast, a large portion of LOX H1 was found in the supernatant of non-treated thylakoids (control in Fig. 5), suggesting a loose association to membranes. However, salt and detergent extractions showed that the fraction of LOX H1 associated with thylakoids was progressively separated from the membrane after treatment with equivalent concentrations of salt and detergent to those required for the extraction of AOS and HPL (not shown). Indeed, a large concentration of salt (1 M) only had a moderate effect in separating LOX H1, AOS, and HPL proteins from the membranes. Moreover, significant fractions of these proteins remained associated to membranes at concentrations of 0.5% Triton X-100 (not shown). In contrast to these enzymes, the portion of AOC associated with thylakoid membranes was immediately extracted after incubation in buffer (Fig. 5).
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Again, LOX H3 could not be detected in chloroplast extracts, suggesting that the protein was labile in the conditions used for chloroplast isolation (not shown).
Subcellular localization of lipoxygenase pathway enzymes using electron microscopy
As shown in the subcellular fractionation experiments above, significant amounts of the oxylipin pathway enzymes are detected within chloroplasts isolated from non-wounded plants. However, chloroplast isolation requires mechanical disruption of the tissues that may affect the accumulation and subcellular distribution of these proteins. Therefore, localization of the LOX pathway enzymes using ultrastructural electron microscopy was performed on non-damaged potato leaves. Polyclonal antibodies prepared against each of the recombinant proteins (or raised against an isoform-specific peptide, in the case of LOX H3) were used to localize LOX H1, LOX H3, HPL, AOS, and AOC in ultrathin sections of potato leaf tissue. For all enzymes, association to different degrees with both thylakoid membranes and stroma could be observed (Figs 6, 7), largely consistent with the results obtained by western blotting. Essentially, no labelling was observed outside chloroplasts. Moreover, no specific labelling was observed when the corresponding preimmune sera were used (not shown).
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Quantitation of results from electron microscopy shows that the largest part of both LOX H1 and LOX H3 localizes to thylakoids with a significant percentage of both of them found in the stroma. HPL and AOS localized almost exclusively to the thylakoid membranes, where a large part of AOC was also found associated with membranes (Table 1). As mentioned before, the AOS antibody used does not discriminate between AOS-1 and AOS-2 isoforms.
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Double labelling using a combination of rat and rabbit polyclonal antibodies was used to investigate the co-localization of AOS and HPL, since both enzymes utilize the same substrate. In these experiments, cryosections of isolated chloroplasts were used in order to improve labelling efficiency. Electron microscopy of frozen ultrathin sections shows that both enzymes are simultaneously present in the chloroplast. However, a differential distribution of HPL and AOS enzymes associated with the same thylakoid was clearly noticed. Areas labelled with AOS antibodies, mainly stacked grana, were essentially free of any HPL labelling that was, in most cases, associated with grana margins and stroma thylakoids (Fig. 8). This differential distribution of AOS and HPL between stacked grana and grana margins is essentially identical to that observed in the single labelling experiments shown in Fig. 7, indicating that it is independent of the fixation techniques used. Interestingly, LOX H1 exhibited an association with the non-appressed part of the thylakoid membrane (Fig. 6) identical to HPL, suggesting that a metabolic interaction between the two enzymes may occur.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The 13-lipoxygenase pathway of fatty acid metabolism yields products that are important in plant defence responses (Hildebrand et al., 2000; León et al., 2001; Vancanneyt et al., 2001). The present work provides compelling evidence gathered in a single plant species, the potato, that this synthetic pathway is confined to chloroplasts, and more specifically to thylakoid membranes, from the oxygenation of the unsaturated fatty acids to the production of C6 aldehydes and the JA precursor, OPDA. Moreover, none of the results obtained with different approaches gave an indication in favour of an additional localization of the proteins involved (13-LOX encoded by LOX H1 and LOX H3, AOS-1 and AOS-2, HPL, and AOC) in other subcellular compartments. This study provides for the first time strong experimental support for the association of this branch of oxylipin synthesis to the thylakoid membrane. Thylakoids are thus key sites for oxylipin synthesis, and hence for plant defence responses against pests and pathogens.
In the present work, the specific distribution of these enzymes within the potato chloroplast has been determined by a number of approaches. Transient expression of GFP-tagged proteins has enabled a detailed localization of single-gene products. A patchy fluorescence pattern was observed in chloroplasts that accumulated transiently expressed AOS-1–, AOS-2–, and HPL–GFP fusions. The coincidence of the number of fluorescence spots detected in transformed chloroplasts with the average number of grana present in the chloroplasts of the A. thaliana cells used for transformation, suggested an association of AOS-1, AOS-2 and HPL to thylakoid membranes. The diffuse fluorescence of the AOC-GFP protein suggests that AOC would probably be abundant in the stroma but does not exclude its association to thylakoids. The pattern of fluorescence observed with the GFP fusions of both LOX H1 and LOX H3 was a mixture of those observed for AOC, and AOS and HPL, and suggested that those plastidial enzymes could be both soluble and membrane-bound.
Fluorescence associated with the transiently expressed GFP-fusion proteins was never observed outside chloroplasts. More specifically, comparison of the localization patterns observed with the GFP-tagged oxylipin proteins to the Tic40- and OEP7-GFP fusions excludes the possibility of a confined association of those proteins to the chloroplast envelope.
Immunolocalization by electron microscopy confirmed and extended the results obtained with confocal microscopy. LOX H1 and LOX H3 were detected both in the stroma and thylakoid membranes. In fact, the subcellular distribution patterns exhibited by LOX H1 and LOX H3 were very similar. Antibodies directed against either AOS or HPL mainly immunodecorated grana thylakoids, with only a small percentage of cross-reacting particles found in stromal sections. A significant percentage of AOC was detected close to, or associated with thylakoids. This association could relate to the requirement for a close proximity between AOC and AOS to perform the conversion of the unstable allene oxide to the first cyclic precursor of JA. Chloroplast fractionation and western blotting studies lend further support to the distribution of enzymes determined by electron microscopy. The minor discrepancies in the localization of proteins (especially AOC), as determined by either electron microscopy or subcellular fractionation, are likely to relate to their differential association to the thylakoid membrane. AOS and HPL exhibit a strong association, while AOC is weakly bound to the membrane and a significant fraction of it dissociates during fractionation procedures.
The localization of potato AOS and HPL to thylakoids differs from the results obtained from in vitro import experiments of the tomato proteins (Froehlich et al., 2001). It seems unlikely that potato and tomato possibly exhibit a different suborganellar distribution for AOS and HPL. The different experimental approaches taken, in vitro import versus in vivo localization of endogenous proteins, may help to explain the discrepancies in the results obtained.
As mentioned before, it was also described that chloroplast envelopes were a major location for oxylipin metabolism (Blée and Joyard, 1996). Indeed, in some experiments a minor part of AOS and HPL has been detected in chloroplast envelope fractions that may perhaps represent proteins in transit to their final destination. However, the bulk of these proteins always appears in either the stromal (LOX H1) or the thylakoid (LOX H1, AOS and HPL) fractions. Moreover, high HPL activity is detected in thylakoid fractions that are essentially devoid of envelope membranes, as determined by the use of several protein markers of the inner and outer chloroplast envelopes. The levels of HPL activity found in thylakoids, determined as cis-3-hexenal and trans-2-hexenal production, are similar to the levels of hydroperoxide consumption and aldehyde formation previously reported for envelope membranes (Blée and Joyard, 1996). In contrast, in the present work, HPL activity in the envelope fractions was below the limits of detection. The use of different fractionation procedures may be responsible for the conflicting results obtained. A recent bioinformatics study suggests the existence within chloroplasts of a system similar to cytosolic vesicular trafficking (Andersson and Sandelius, 2004). Some fractionation procedures may thus result in envelope fractions enriched in envelope-derived vesicles with cargo proteins destined to the thylakoid membrane.
The spatial distribution of enzymes within chloroplasts described in the present work suggests that oxygenation of unsaturated fatty acids and subsequent hydroperoxide processing would proceed at or near the inner plastidial membranes while OPDA formation could, in part, occur in the stroma. It is important to consider in this regard the hydrophobic nature of most intermediates in this metabolic pathway, that probably demand a hydrophobic micro-environment, probably in association with membranes, where reactions may take place. The membrane-bound LOX H1 fraction appears to be as tightly associated to thylakoids as HPL and AOS. The additional presence of LOX H1 in the stroma suggests that localization of LOX H1 may be a dynamic process influenced by external, so far uncharacterized stimuli, which may direct LOX H1 towards the membrane for interaction with its fatty acid substrate. Reversible association with membranes has already been described for soybean LOX L-1 and other enzymes utilizing lipid substrates (Tatulian et al., 1998). The assembly of the LOX metabolic pathway is likely to depend on migration of proteins (LOX and AOC) from the stroma to the membrane rather than to changes of localization of integral membrane proteins such as HPL and AOS.
Double-labelling with the different antibodies (only shown for AOS and HPL) shows for the first time that individual chloroplasts contain all enzymes for the divergent branches of the lipoxygenase pathway, in which two enzymatic steps for alternative routing are evident. Firstly, unsaturated fatty acids may be substrates of either LOX H1 or LOX H3, which, however, will yield identical 13-hydroperoxide products. All evidence gathered in the present work suggests that both enzymes share the same location and may thus have access to the same substrates. Secondly, their 13-hydroperoxides can be used by either AOS or HPL, which are both associated to thylakoids. In this case, however, co-localization experiments by immuno-electromicroscopy show that AOS and HPL may be physically separated. Domains within grana thylakoids have been described (Albertsson, 2001), in which photosystem I and II are laterally segregated. While photosystem II is essentially restricted to appressed core of grana (where HPL is detected), photosystem I is distributed in grana margins, end grana membranes and stroma lamellae, where most of AOS labelling is localized. Although the significance of this observation is as yet uncertain, it may, however, be a determinant of either the accessibility of the 13-hydroperoxide substrates to these enzymes in particular situations or the efficiency of the enzymatic reaction. This choice in utilization of 13-hydroperoxides is likely to determine the specificity of the defence response to be mounted. Indeed, the use of 13-hydroperoxides by AOS leads to activation of JA-responsive genes, an essential component of responses to chewing insect pests (Royo et al., 1999). If, on the contrary, 13-hydroperoxides are used by HPL, C6 aldehydes are formed that are deterrents for sucking insects (Vancanneyt et al., 2001). Data from transgenic plants with either of these routes blocked suggested that HPL could not use 13-hydroperoxides made by LOX H3 and, on the other hand, 13-hydroperoxides made by LOX H1 would not replace those made by LOX H3 in the activation of JA-responsive genes (Royo et al., 1999; Vancanneyt et al., 2001). The present work shows that wound-induced accumulation of LOX H3, AOS, and AOC may, in part, be responsible for the observed partitioning of 13-hydroperoxides, in that LOX H3 would not be available in non-damaged LOX H1-co-suppressed plants to provide 13-hydroperoxides for C6 aldehyde formation. However, AOS should be able to use 13-hydroperoxides made by LOX H1, which is still a relatively abundant protein in wounded LOX H3 antisense plants. Hence, a metabolic compartmentalization that would restrict access of AOS to LOX H1-made 13-hydroperoxides prevails as a distinct possibility to explain these results, that may relate to the physical separation of AOS and HPL mentioned above. Interestingly, both LOX H1 and HPL largely localize to non-appressed grana.
On the AOS branch of the lipoxygenase pathway, the OPDA product of AOC has to be transported to peroxisomes where it will undergo ß-oxidation to yield JA (Theodolou et al., 2005). Strong evidence is now provided to demonstrate that a large part of AOC resides in the stroma. An association of AOC with the external plastidial envelope, as could be expected for a directional transport of its OPDA product out of the chloroplast into peroxisomes, has never been observed (Ziegler et al., 2000; T Farmaki, unpublished results). A physical association between these two enzymatic activities was suggested (Ziegler et al., 2000; León and Sánchez-Serrano, 1999) on the basis of the very short half-life of the allene oxide intermediate in aqueous solution that would perhaps require the proximity of the sequential enzyme activities for an efficient reaction. As argued above, wound-dependent migration of the soluble stromal AOC to the membrane where AOS resides seems a likely possibility. However, a minor part of AOS also localizes to stroma, and the membrane bound/stromal AOS protein ratio may well be altered upon wounding if this differential localization has a functional significance. These possibilities are quite pertinent for the understanding of the channelling of metabolic intermediates in the LOX pathway, and are currently under investigation.
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Acknowledgements |
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We gratefully acknowledge Pilar Paredes for excellent technical assistance. We want to thank Professor Kenneth Keegstra for the Toc75, Tic40, and Tic110 antibodies, Professor Klaus Kloppstech for the LHC II antibody, and Monsanto Co, St Louis MO, for providing the pMON30063 vector. We also wish to thank Dr Maite Rejas and the Electron Microscopy Service from the Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, for assistance in this work. Dr Julio Salinas made very useful comments and suggestions on the manuscript. Financial support was provided by the Spanish Comisión Interministerial de Ciencia y Tecnología grants BIO99-1225 and BIO2002-03926 (to JJSS). TF was supported by a Marie Curie Individual Fellowship from the Commission of European Communities.
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Footnotes |
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Present address: Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Avenida Padre García Tejero 4, 41012 Sevilla, Spain. 
Present address: Bayer BioScience NV, Technologiepark 38, B-9052 Gent, Belgium.
Present address: Instituto de Biología Molecular y Celular de Plantas. Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas. 46022 Valencia, Spain.
Database accession: Allene oxide cyclase cDNA, GenBank accession number AY135641; allene oxide synthase 2 cDNA, GenBank accession number AY135640.
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Abbreviations |
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AOC, allene oxide cyclase; AOS, allene oxide synthase; HPL, hydroperoxide lyase; LOX, lipoxygenase; GFP, green fluorescent protein; JA, jasmonic acid.
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