JXB Advance Access originally published online on September 12, 2006
Journal of Experimental Botany 2006 57(14):3553-3562; doi:10.1093/jxb/erl108
RESEARCH PAPER |
Sethoxydim affects lipid synthesis and acetyl-CoA carboxylase activity in soybean
1Physiologie Végétale/LBPO, Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumédienne, BP 39, El Alia, Bab Ezzouar, Alger, Algérie
2Institut de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris-Sud, F-91405 Orsay, France
3Université Pierre et Marie Curie-Paris 6, FRE 2846, PCMP, Ivry-sur-Seine, F-94200 France
* To whom correspondence should be addressed. E-mail: depaepe{at}ibp.u-psud.fr
Received 4 May 2006; Accepted 3 July 2006
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Abstract |
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With rare exceptions, dicot plastids have been reported to contain only a multisubunit (prokaryotic) form of acetyl-coA carboxylase (ACCase), the first committed step of lipid biosynthesis. The sensitivity of most monocots to cyclohexanediones (CHDs) such as sethoxydim, has been shown to be associated with the presence in their plastids of a multifunctional (eukaryotic) form of ACCase. Little is known about the effects of sethoxydim on lipid metabolism and ACCase activity in dicots. Here it is shown that foliar lipid biosynthesis is differentially affected by the herbicide treatment in two dicot species, Nicotiana sylvestris (wild tobacco) and Glycine max (soybean). In N. sylvestris, the total lipid content of neoformed leaves harvested 2 weeks after the sethoxydim treatment was unaffected by doses of up to 10–3 M sethoxydim. In soybean, lipid content decreased by 45% when 10–5 M sethoxydim was used, and this was associated with a 30% reduction in fatty acid synthesis activity. ACCase activity of soybean plastidial preparations was 60% reduced in the presence of sethoxydim, whereas that of N. sylvestris was unaffected. Finally, the presence of a biotinylated 220 kDa polypeptide, corresponding in size to multifunctional ACCase, was observed in soybean plastids. Possible relationships between sensitivity of plastidial soybean ACCase towards sethoxydim, plastidial protein content, and altered de novo lipid biosynthesis in herbicide-treated plants are discussed.
Key words: Acetyl-CoA carboxylase (ACCase), fatty acid synthesis, plant lipid metabolism, sethoxydim
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Introduction |
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In plants, de novo fatty acid biosynthesis mainly takes place in the plastidial compartment (Ohlrogge and Browse, 1995). Two enzyme systems are required for fatty acid formation: acetyl-CoA carboxylase (ACCase, EC 6.4.1.2 [EC] ) and fatty acid synthase. The first step in the pathway from acetyl-CoA to long-chain fatty acids is the production of malonyl-CoA, catalysed by ACCase, of which two forms have been identified in plants (Egli et al., 1993; Alban et al., 1994; Konishi and Sasaki, 1994; Sasaki and Nagano, 2004). The multisubunit (MS) ACCase (prokaryotic type) present in plastids of all plants, except Poaceae and Geraniaceae, is composed of four non-identical polypeptides which catalyse two reactions: the biotin carboxylase (BC) subunit catalyses the ATP-dependent carboxylation of the biotinyl moeity on biotin carboxyl carrier protein (BCCP), and the
and ß carboxyltransferase (CT) subunits catalyse the transfer of activated carboxyl groups from BCCP to acetyl-CoA to form malonyl-CoA. The MS ACCase subunits are encoded by nuclear genes, except the CT subunit which is encoded by the plastidial genome (Konishi et al., 1996). The MS complex is involved in de novo fatty acid synthesis. The multifunctional (MF) ACCase, consisting of a single 220–240 kDa polypeptide with BCCP, BC, and CT domains, is nuclear encoded. In all plants, it is assigned to the cytosolic compartment where it is involved in fatty acid elongation and flavonoid biosynthesis. Poaceae and Geraniaceae contain MF ACCase instead of MS ACCase in their plastids (Christoffers and Holtum, 2000). Another exception is Brassica napus, the plastids of which have been reported to contain both MS and MF types of ACCase (Schulte et al., 1997). The MF ACCase of Poaceae plastids is known to be the target of two chemically distinct classes of inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones (CHDs), commonly referred to as graminicides, while the MS ACCase is much less sensitive (Sammons et al., 1988; Rendina et al., 1990; Konishi and Sasaki, 1994). Sensitivity determinants are located in the CT domain of the MF ACCase (Nikolskaya et al., 1999; Délye et al., 2002, 2003b). Resistance towards sethoxydim in some grass species, such as Lolium rigidum (Zagnitko et al., 2001), Avena fatua (Christoffers et al., 2002), Setaria italica (Delye et al., 2002), Festuca rubra, and Alopecurus myosuroides mutant populations (Delye et al. 2003a), is associated with the substitution of a leucine by an isoleucine in the CT domain of MF ACCase. The inhibition of ACCase activity by graminicides results in severe growth defects leading to plant death in <2 weeks (Walker et al., 1988).
In this work, the effects of a CHD class herbicide, sethoxydim, at various doses (10–5–10–2 M) on the in planta lipid metabolism of two dicot species, Nicotiana sylvestris (wild tobacco) and Glycine max (soybean) were compared. It is shown that, in contrast to tobacco, lipid content and fatty acid synthesis were severely affected in newly formed soybean leaves having developed during 2 weeks after the herbicide treatment. Moreover, ACCase activity was strongly reduced in plastidial soybean preparations treated with the herbicide.
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Materials and methods |
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Plant growth conditions and herbicide treatment
Soybean (G. max L. var. Weber) and N. sylvestris plants were grown in a greenhouse under controlled conditions (16 h day at 25 °C and 8 h night at 18 °C). Sethoxydim (2-[1-(ethoxyiamino)]butyl-5-[2-ethyl-thio propyl]-3-hydroxy-2-cyclohexen-1one, Nippon-Soda) was dissolved in water and diluted before application. A 50 ml aliquot of different concentrations from 10–5 to 10–2 M was sprayed onto young G. max plantlets (first leaf-stage) or 2-month-old N. sylvestris plants (rosette stage). All in planta analyses were performed on the leaves having developed during the 2 weeks after the herbicide treatment, referred to hereafter as ‘neoformed leaves’. Neoformed leaves of untreated plants were used as a control. The effects of the herbicide later in development were not examined. This is in contrast to field treatments of soybean, where plants were sprayed at the leaf 3–6 stage (V3–V6 growth stages), and the herbicide effect examined on vegetative and reproductive development and on yield (Nelson and Renner, 2001).
Lipid analysis
Harvested leaves were fixed in boiling water. Lipids were extracted by methanol:chloroform:water (1:1:1, by vol.) (Bligh and Dyer, 1959). Lipid classes were separated by thin-layer chromatography (TLC) according to Lepage (1967). For fatty acid analysis, aliquots of the total lipid extract or spots corresponding to lipid classes separated by TLC were methylated and analysed by capillary gas chromatography (GC), isothermally at 170 °C, using a Girdel apparatus equipped with a 50 m long, 0.25 mm diameter carbowax column. Heptadecanoate was added as a standard for quantitative determination of fatty acid contents.
Labelling experiments
A 60 µl aliquot of a solution of sodium [1-14C]acetate (2.1 GBq mmol–1, Amersham) was placed in situ on to the upper surface of neoformed leaves of control or treated plants (in total six leaves of soybean and three leaves of N. sylvestris). The administered radioactivity per leaf corresponded to 16 µmol of sodium acetate for soybean and 32 µmol for N. sylvestris. Total lipid was extracted according to Bligh and Dyer (1959). Lipid classes were separated by TLC, and the radioactivity of total lipid and lipid classes was counted by liquid scintillation spectrometry.
Isolation of purified chloroplasts
Intact soybean and N. sylvestris chloroplasts were purified according to Douce et al. (1990) and lysed by osmotic shock. The osmotic pressure was adjusted with buffer containing 0.2 M Tricine–KOH pH 8.0, 0.6 M glycerol, 2 mM dithiothreitol (DTT), and the volume was completed with the same buffer diluted 2-fold. The soluble protein fraction was separated from membranes by centrifugation at 15 000 g for 15 min and assayed for ACCase activity.
Assay for in vitro ACCase activity
ACCase activity was quantified by measuring the incorporation of [1-14C]NaHCO3 (Amersham) into the chloroplastic soluble protein fraction (Alban et al., 1994). The reaction mixture (300 µl final volume) contained 50 mM HEPES, pH 8.0, 1 mM ATP, 2.5 mM MgCl2, 20 mM KCl, 0.5 mM DTT, 3 Ci ml–1 [1-14C]NaHCO3, 0.4 mM acetyl-CoA, 100 µg of protein, and 5 µl of sethoxydim solutions at different concentrations.
The reaction was initiated by the addition of acetyl-CoA. The reaction mixture was placed at 30 °C for 10 min. Aliquots of the reaction mixture (150 µl) were mixed vigorously with 40 µl of 12 N HCl to stop the reaction. The solution was then dried under N2 and the acid-stable radioactivity was quantified in a liquid scintillation counter. Duplicate assays without acetyl-CoA were run as controls.
Total protein extraction
Leaf samples (100 mg) were ground in liquid nitrogen and total proteins were extracted in 0.3 ml of 0.1 M TRIS–HCl pH 8.1, 10% sucrose, 0.05% ß-mercaptoethanol. The extract was centrifuged for 10 min at 20 000 g to eliminate insoluble material, and protein content was determined according to Bradford (1976).
Electrophoretic analysis and western blotting
SDS–PAGE was performed at room temperature using 9% polyacrylamide gels (20 µg protein per lane). After electrophoresis, the polypeptides were transferred electrophoretically on to nitrocellulose membranes essentially according to Towbin et al. (1979). Biotin-associated polypeptides were identified using horseradish peroxidase-labelled streptavidin (Baldet et al., 1992).
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Results |
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Sethoxydim affects growth of Glycine max plantlets
Growth of soybean plantlets was markedly affected by sethoxydim treatment, even at a concentration of 10–5 M (Fig. 1). After 2 weeks, treated leaves were fully necrotic, whereas neoformed leaves were chlorotic (Fig. 1B). When higher doses were applied (10–2 M), the neoformed leaves developed poorly (Fig. 1C). When compared with control plants (Fig. 1A), the chlorophyll content of neoformed leaves decreased from 2.12 mg g–1 FW in control plant to 1.40 mg g–1 FW for the 10–5 M sethoxydim treatment and to 1.16 mg g–1 FW for the 10–2 M treatment. In contrast, N. sylvestris leaves were not affected by sethoxydim until the application of 10–3 M, and no decrease in chlorophyll content was observed in neoformed leaves. However, at the 10–2 M concentration, chlorosis and a 38% decrease in chlorophyll content were found in treated leaves when compared with the control plants.
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Sethoxydim differentially affects polar lipid content in Glycine max and N. sylvestris leaves
The effect of sethoxydim on the polar lipid content of neoformed leaves harvested 2 weeks after the herbicide treatment is shown in Fig. 2. In soybean, the lipid content decreased from 6.6 mg g–1 FW in control leaves to 3.6 mg g–1 FW in plants treated with 10–5–10–2 M sethoxydim (Fig. 2A). In N. sylvestris, the treatment had no effect on polar lipid content between 10–5 and 10–3 M sethoxydim. However, when 10–2 M sethoxydim was used, the lipid content decreased by
25% (Fig. 2B).
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Leaf fatty acid composition is affected by sethoxydim in soybean only
Higher plants possess two different pathways of glycerolipid biosynthesis. The prokaryotic pathway is exclusively located in the chloroplasts, while the eukaryotic pathway involves both the chloroplastic compartment and the endoplasmic reticulum (Ohlrogge and Browse, 1995). Plants which contain glycolipids with diacylglycerol (DAG) backbones synthesized via both pathways contain hexadecatrienoic acid (C16:3) and linolenic acid (C18:3) and are called C16:3 plants, whereas C18:3 plants have only the eukaryotic pathway and are characterized by a high linolenic acid content.
The fatty acid composition analysis of soybean and N. sylvestris leaves reflected differences in their lipid biosynthesis pathways. As expected, glycerolipids of N. sylvestris, a C16:3 species, contained 2.6% hexadecatrienoic acid and 48.1% linolenic acid, whilst lipids of soybean, a C18:3 species, contained 67% linolenic acid (Table 1).
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The fatty acid composition of N. sylvestris neoformed leaves was relatively unaffected by sethoxydim, whatever the concentration used, whereas the fatty acid composition of soybean leaves was markedly modified by the herbicide treatment. These changes were independent of the concentration used. A marked decrease in linolenic acid was noted, from 67% in control to 50% in treated plants. In contrast, a significant increase in the proportion of C16:0 fatty acids was observed, from 6.7% to 20% (Table 1). In terms of total lipid content, the C18 fatty acids declined from 6.0 mg g–1 FW to 2.4 mg g–1 FW, whereas the C16 fatty acid content slightly increased.
Sethoxydim essentially affects chloroplastic lipids in soybean
The effect of sethoxydim on lipid class composition was examined in both species. In soybean, the chloroplastic lipids of treated leaves were affected, with a large decrease in galactolipids (Fig. 3). The sethoxydim treatment induced a reduction in monogalactosyl diglycerol (MGDG) (60%), digalactosyl diglycerol (DGDG) (40%), and phosphatidylglycerol (PG) (20%). The content in extrachloroplastic phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) was not significantly affected (Fig. 3A).
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In N. sylvestris leaves, there were no significant changes in the lipid classes following the application of 10–5 M sethoxydim (data not shown). However, at 10–3 M, a 30% reduction of PG was noted, while MGDG, the major chloroplast lipid, was unaffected by the treatment. Extrachloroplastic phospholipid content remained unchanged (Fig. 3B).
Sethoxydim affects fatty acid synthesis in soybean
To examine the effect of sethoxydim on fatty acid synthesis, neoformed leaves of control plants and plants treated with 10–3 M sethoxydim were radiolabelled with sodium [1-14C]acetate. In both species, the labelling of total lipids reached an equilibrium after 6 h (Table 2). In untreated plants, the labelling of total fatty acids was >2-fold higher in soybean than in N. sylvestris, 47 µmol versus 21 µmol g–1 FW. Acetate uptake may be species- or age-dependent, as previously reported in pea (Hellgren and Sandelius, 2001), but it is clear that the herbicide treatment had a differential effect between the two species. In soybean, it led to an 30% reduction in [1-14C]acetate incorporation. On the other hand, sethoxydim induced a 60% increase in lipid labelling of N. sylvestris leaves (Table 2).
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The distribution of radioactivity in the major lipid classes was examined in treated and untreated plants (Fig. 4). In soybean leaves, the labelling of all lipid classes, except PC and PE, was lowered by the herbicide treatment (Fig. 4A). After 6 h, MGDG labelling was reduced by 64%, DGDG labelling by 25%, and PG labelling by 53%. In contrast, in N. sylvestris leaves, the sethoxydim treatment induced an increase in acetate incorporation in most classes of both chloroplastic and extrachloroplastic lipids. The radioactivity incorporated into MGDG increased by 86%, into DGDG by 30%, and into PG by >100% (Fig. 4B).
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Taken together, lipid content, fatty acid analyses, and acetate incorporation measurements strongly suggest that fatty acid synthesis is affected by sethoxydim in soybean, resulting in the exhaustion of chloroplastic lipids. Some compensatory mechanisms seemed to take place at the level of the extraplastidial compartments. In N. sylvestris, the herbicide treatment resulted in an increased fatty acid synthesis, although no changes in global lipid content could be observed.
ACCase activity of soybean chloroplastic preparations is affected by sethoxydim
Since it has been demonstrated that ACCase exerts a strong flux control over lipid synthesis (Page et al., 1994; Ohlrogge and Jaworsky, 1997), the hypothesis was tested that impairment of lipid content and fatty acid synthesis in sethoxydim-treated soybean plants might be due to a susceptibility of plastidial ACCase towards the herbicide. The in vitro activity of ACCase was assayed on purified chloroplasts from control plants in the presence of different sethoxydim concentrations, from 10–5 to 10–2 M (Fig. 5). At all concentrations tested, the treatment had no effect on the ACCase activity of N. sylvestris chloroplasts (Fig. 5B). In contrast, in soybean, each herbicide concentration induced an 60% reduction in chloroplastic ACCase activity (Fig. 5A).
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Patterns of biotinylated leaf and chloroplastic proteins are different in N. sylvestris and soybean
Experiments were carried out to determine whether differences in ACCase composition could be detected between N. sylvestris and soybean. As the ACCase BCCP domain is biotinylated, total leaf and chloroplast proteins were compared by western analysis using streptavidin, according to Baldet et al. (1992).
In the total soluble protein extracts from leaves and plastids of N. sylvestris, a major 38 kDa polypeptide corresponding in size to the BCCP subunit of the MS enzyme was observed (Fig. 6). A protein in the 75–80 kDa range that could correspond to mitochondrial methylcrotonyl-CoA carboxylase (Baldet et al., 1992; Alban et al., 1993) gave a weaker signal mainly in total leaf extracts. In addition, a signal at 220 kDa was clearly detectable in total leaf proteins and this could correspond to the cytosolic MF ACCase, the molecular mass of which is 220–240 kDa. Such a high molecular mass signal was not detected in the plastidial fractions, in good agreement with the general extrachloroplastic location of MF ACCase in dicots.
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The biotinylated profiles of soybean total and chloroplastic proteins showed several differences compared with those of N. sylvestris. First, two polypeptides in the 38–40 kDa range were observed, suggesting the presence of at least two different BCCP isoforms. Similar observations have been reported by Reverdatto et al. (1999) in soybean, and multiple tissue-specific BCCP subunits have been described in a number of plant species (Thelen et al., 2001). Secondly, a polypeptide in the 90 kDa range and diffuse signals in the 180 kDa range were observed in both total and plastidial extracts that could correspond to non-dissociated BC–BCCP subcomplexes, as already proposed for soybean by Reverdatto et al. (1999). Finally, although no obvious signal at
220–240 kDa was visible in total leaf proteins, a band in this molecular weight range was clearly visible in chloroplastic extracts, which might either correspond to an MF ACCase isoform or to the aggregation of MS ACCase subunits.
Sethoxydim does not affect accumulation of BCCP subunits
Accumulation of BCCP in soybean total proteins from control plants and from plants treated with 10–5 and 10–2 M sethoxydim was analysed by western analysis using streptavidin (Fig. 7). No significant changes in BCCP amounts were observed following sexothydim treatment, suggesting that the herbicide did not affect the accumulation of MS ACCase.
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Discussion |
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Sethoxydim is a post-emergence herbicide used to eliminate weeds in dicotyledonous cultures. It is absorbed by the leaves and transported throughout the phloem towards the apical meristem. Systemic herbicides require from several days to a few weeks to move throughout the vascular system of a treated plant. In monocots, necrotic zones develop in neoformed leaves, resulting in growth inhibition and cell death in 1 or 2 weeks. In dicots, effects of post-emergence herbicide have been reported under field conditions (Griffin and Habetz, 1989; Hart and Roskamp, 1998), but plants most often recover from injury, depending on growth conditions and seasons (Kapusta et al., 1986). Both leaf area index and plant height were affected in soybean up to 52 d after treatment with tank mixtures including sethoxydim (Nelson and Renner, 2001). However, such deleterious effects have not been reported following application of sethoxydim alone.
Surprisingly, in the present greenhouse experimental conditions, a marked phenotypic response towards 10–5 M sethoxydim, a dose often used in the field, was observed in soybean, whilst N. sylvestris leaves presented symptoms only for the higher dose (10–2 M) of the herbicide. Necrotic areas appeared on soybean treated leaves, whereas the first neoformed leaves, harvested 2 weeks after the treatment, showed reduced development and chlorotic symptoms. These striking differences between greenhouse- and field-grown conditions could be due to a combination of different effects. First, in the present experiments, soybean plants were sprayed at the first leaf-stage, whilst in the field, treatments were applied to plants at an older developmental stage, generally at the leaf 3–5 stages (V3–V5, Nelson and Renner, 2001), and older plants could be less sensitive.
Moreover, the herbicide retention time may be increased in young greenhouse-grown plants, due to lower catabolic and detoxification mechanisms since cytochrome P450 and glutathione transferase activities are both dependent upon age and environment. In particular, it has been shown that light accelerates the elimination of the herbicide under field conditions (reviewed in Werck-Reichhart et al., 2000; Delye, 2005). It is also likely that the graminicide half-life in the soil is higher in the greenhouse than under field conditions, due to the artificial soil composition, absence of rainfall, and microbial remediation.
Whatever the exact causes of the sethoxydim toxicicity against the soybean in the present experiments, these effects were associated with a 45% reduction in total lipid content and a 30% reduction in lipid synthesis of neoformed leaves of treated plants, when compared with control plants. In contrast, lipid synthesis was not affected in N. sylvestris. Little information is available about the possible effects of graminicides on in planta lipid metabolism in dicots. Pea leaves have been reported to be insensitive to the AAP fluazifop (Walker et al., 1988, 1989). Differential effects of graminicides on isolated dicot chloroplasts have been reported. For example, sethoxydim had no effect on pea chloroplasts (Burton et al., 1987). In contrast, a 50% reduction in the incorporation of [1-14C]acetate into isolated spinach chloroplasts, treated with 10–4 M diclofop (an aryloxyphenoxypropionic herbicide) has been reported, whereas pea chloroplasts were less sensitive (Kobek et al. 1987).
It is generally accepted that the specific effect of sethoxydim on lipid biosynthesis in monocots is caused by the presence of a sensitive plastidial MF ACCase, whereas, with the possible exception of B. napus, it is believed that dicots only possess a resistant MS form (see Introduction). Unexpectedly, it was observed that the ACCase activity of isolated soybean chloroplasts was 60% inhibited by the addition of sethoxydim. The lack of a dose dependence in the range of concentrations used suggests the presence of two different ACCase activities in soybean chloroplasts: one activity that is fully sensible to the lower dose used, i.e. 10–5 M, and one resistant up to the higher dose, i.e. 10–2 M.
In contrast, ACCase activity of isolated N. sylvestris chloroplasts was unaffected by the herbicide. Tests were performed to determine whether the differences between the two species could be related to differences in leaf plastidial ACCase composition. Only one abundant 35 kDa biotinylated polypeptide, corresponding in size to the BCCP polypeptide, could be detected in tobacco plastid preparations, whilst in soybean two such polypeptides were observed, suggesting the presence of at least two plastid MS ACCase isoforms. The presence of several BCCP polypeptides has been described in a number of plant species, including soybean seedlings (Elborough et al., 1996; Reverdatto et al., 1999). In B. napus, one BCCP subunit is expressed in all tissues, whereas the second is only expressed in reproductive organs (Thelen et al., 2001). Whether the two leaf soybean subunits are cell specific or respond to different stimuli is not known. However, it is very unlikely that one of them could confer resistance to sethoxydim, as in all cases reported so far the determinant of the resistance was found to be located in the MF plastidic ACCase (Nikolskaya et al., 1999; Zagnitko et al., 2001). Therefore, the susceptibility of soybean chloroplastic ACCase activity towards the herbicide would be best explained by the presence of an MF ACCase isoform in plastids. In good agreement with this, a biotinylated protein was detected in the 220 kDa range in soybean plastidial extracts, that cannot be explained by a contamination of cytosolic MF ACCase as this protein was not detectable in total leaf extracts. In contrast, a 220 kDa biotinylated protein was clearly visible in N. sylvestris total leaf proteins but not in plastidial extracts. Although the possibility cannot be excluded that the 220 kDa band of soybean plastids might correspond to the aggregation of MS ACCase subunits, our results are in agreement with the proposition that soybean chloroplasts contain both the MS and MF forms of the enzyme. In contrast, our experimental conditions did not provide evidence for the presence of a plastidial MF ACCase in N. sylvestris. However, tobacco plants have been used at an older stage than soybean (2 months old instead of 1 month old), and stage dependence may be considered, as discussed before. Whatever the case, a good correlation was observed between the presence of the 220 kDa polypeptide in plastids and inhibition of plastidial ACCase activity and of lipid biosynthesis.
The presence of a MF ACCase in dicot plastids has already been proposed for chickpea (Giménez-Espinosa et al., 1999) and for B. napus using a genomic strategy (Schulte et al., 1997). In support of the latter result, the B. napus MF ACCase2 gene possesses a putative plastidial targeting sequence, with a prediction probability score of 0.9 using Target P (Emanuelsson et al., 2000). Interestingly, sethoxydim induces a 30–40% decrease in both ACCase activity and fatty acid synthesis in B. napus (A Belkebir, unpublished results). The Arabidopsis genome also contains two MF ACCase genes (Nikolau et al., 2003), one of which is predicted to encode a plastid-located enzyme (Target P prediction of 0.8). Such in silico analyses suggest that an MF ACCase could be present in the plastids of a number of dicot species, as discussed in Nikolau et al. (2003). However, to the best of our knowledge, no MF ACCase gene with a putative chloroplastic transit peptide presequence has been characterized so far in soybean.
Whether the putative presence of an MF ACCase in soybean chloroplasts might be directly responsible for the observed 60% decrease in plastidial ACCase activity remains to be demonstrated however. Based on the intensity of the 38 kDa and 220 kDa bands, the putative chloroplast-localized MF enzyme seems to be present in much smaller amounts than the MS ACCase, but the difference might be caused in part by a low transfer efficiency of high molecular weight polypeptides. Hence, if the hypothesis of the simultaneous presence of MS and MF ACCase enzymes in soybean plastids is correct, the relative contribution of each isoform to total plastidial ACCase activity remains to be determined. The balance between both activities could be age-dependent, and differ markedly in plants at the 3–5 leaf-stage that are usually sprayed in the field, and in the one leaf-stage plants grown in the greenhouse and used in this study. It has been shown that the activity of MS ACCase is regulated by transcript amounts and editing of the plastid-encoded AccD gene (Madoka et al., 2002), and by post-translational modifications, such as phosphorylation (Savage and Ohlrogge, 1999) and light-driven redox changes (Kozaki and Sazaki, 1999; Kozaki et al., 2001). Thus, in addition to impaired detoxification mechanisms discussed before, changes in relative MF and MS ACCase amounts and/or activities in plastids might contribute to the differential response of sethoxydim-treated soybean plants grown in the field with respect to the present experimental conditions.
Because the cytosolic MF ACCases are relatively insensitive to graminicides, they are not considered to play a role in herbicide toxicity (reviewed in Délye, 2005). However, a range in tolerance of cytosolic MF ACCase to various herbicides has been reported in dicots (Alban et al., 1994; Giménez-Espinosa et al., 1999). Antisense to the MF ACCase cytosolic gene results in lower lipid content in B. napus (Slabas et al., 2002), indicating cross-talk between components of lipid metabolism. Therefore, the sensitivity of cytosolic soybean ACCase to sethoxydim might also affect the lipid content. Moreover, besides a direct inhibition of MF ACCase activity, additional effects of sethoxydim on lipid content have to be considered. Indeed, the herbicide treatment affected fatty acid composition, as seen by an increase in the level of saturation of total fatty acids and a decrease in polyunsaturated fatty acids. The percentage of palmitic acid (C16:0) significantly increased in treated plants when compared with controls. Moreover, the herbicide treatment essentially affected the chloroplastic compartment, with a strong reduction in MGDG, DGDG, and PG contents, while extrachloroplastic lipids were less affected. The diminution in linolenic acid-rich galactolipids can explain the observed decrease in linolenic acid content. A drift in chloroplastic lipid content is often observed under different stress conditions, such as norflurazon treatment (Abrous et al., 1998), heat shock (Aid et al., 1998), and drought (Benhassaine-Kesri et al., 2002). Finally, membrane peroxidation, that has been recently reported to occur following graminicide treatment, could also contribute to changes in lipid composition of chloroplastic membranes (Shimabukuro et al., 2001; Luo et al., 2004). Thus, it is likely that an additional stress response on membrane chloroplastic lipids could be superposed on a direct effect of sethoxydim on ACCase activity.
In conclusion, the results show strong effects of low doses of cyclohexanediones on lipid synthesis and chloroplastic lipid content in young soybean plants. The sensitivity of plastidial ACCase to sethoxydim and the presence of a 220 kDa biotinylated polypeptide in soybean plastids provide a biochemical indication for the possible presence of two ACCase isoforms, one resistant (MS) and one sensitive (MF), in soybean leaf chloroplasts.
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Acknowledgements |
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This work was supported by the French CNRS and Ministry of Research, the Algerian Ministry of Research, and by financial support from CMEP MDU 460 to AB. We wish to thank Michael Hodges (IBP, UPS Orsay) for carefully reading the manuscript and R Boyer (IBP) for the photographic artwork.
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Abbreviations |
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16:0, palmitic acid; 16:1c,
7 hexadecenoic acid; 16:2, 7,10 hexadecadienoic acid; 16:3, 7,10,15 hexadecatrienoic acid; 18:0, stearic acid; 18:1, 9 octadecenoic acid (oleic acid); 18:2, 9,12 octadecadienoic acid (linoleic acid); 18:3, 9,12,15 octadecatrienoic acid (linolenic acid); ACCase, acetyl-CoA carboxylase; APP, aryloxyphenoxypropionate; BC, biotin carboxylase; BCCP, biotin carboxyl carrier protein; CHD, cyclohexanedione; CT, carboxyltransferase; DGDG, digalactosyl diglycerol; FW, fresh weight; GC, gas chromatography; MF, multifunctional; MGDG, monogalactosyl diglycerol; MS, multisubunit; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; TLC, thin-layer chromatography. |
References |
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Abrous O, Benhassaine-Kesri G, Tremolières A, Mazliak P. (1998) Effect of Norflurazon on lipid metabolism in soya seedlings. Phytochemistry 49:979–985.[CrossRef]
Aid F, Benhassaine-Kesri G, Demandre C, Mazliak P. (1998) Modification of the biosynthesis of rape lipid molecular species by heat shock. Phytochemistry 47:1195–1200.[CrossRef]
Alban C, Baldet P, Axiotis S, Douce R. (1993) Purification and characterization of 3-methylcrotonyl-coenzyme A carboxylase from higher plant mitochondria. Plant Physiology 102:957–965.[Abstract]
Alban C, Baldet P, Douce R. (1994) Localisation and characterisation of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides. Biochemical Journal 300:557–565.
Baldet P, Alban C, Axiotis S, Douce R. (1992) Characterization of biotin and 3-methyl-crotonyl-coenzyme A carboxylase in higher plant mitochondria. Plant Physiology 99:450–455.
Benhassaine-Kesri G, Aid F, Demandre C, Kader JC, Mazliak P. (2002) Drought stress affects chloroplast lipid metabolism in rape (Brassica napus) leaves. Physiologia Plantarum 115:221–227.[CrossRef][Medline]
Bligh EG and Dyer WJ. (1959) A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37:103–106.
Bradford MM. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72:248–254.[CrossRef][ISI][Medline]
Burton JD, Gronwald JW, Somers DA, Connelly JA, Gengenbach BG, Wyse DL. (1987) Inhibition of plant acetyl-coenzyme A carboxylase by the herbicides sethoxydim and haloxyfop. Biochemical and Biophysical Research Communications 148:1039–1044.[CrossRef][ISI][Medline]
Christoffers MJ, Berg ML, Messersmith CG. (2002) An isoleucine to leucine mutation in acetyl-CoA carboxylase confers herbicide resistance in wild oat. Genome 45:1049–1056.[Medline]
Christoffers JT and Holtum JAM. (2000) Dicotyledons lacking the multisubunit form of the herbicide target enzyme acetyl-coenzyme A carboxylase may be restricted to the family Geraniaceae. Australian Journal of Plant Physiology 27:845–850.
Délye C. (2005) Weed resistance to acetyl coenzyme A carboxylase inhibitors: an update. Weed Science 53:728–746.[CrossRef]
Délye C, Straub C, Matéjicek A, Michel S. (2003a) Multiple origins for black-grass (Alopecurus myosuroides Huds) target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase. Pest Manadgement Science 60:35–41.
Délye C, Wang T, Darmency H. (2002) An isoleucine–leucine substitution in chloroplast acetyl-CoA carboxylase from green foxtail (Setaria viridis L. Beauv.) is responsible for resistance to the cyclohexanedione herbicide sethoxydim. Planta 214:421–427.[CrossRef][ISI][Medline]
Délye C, Zhang XQ, Chalopin C, Michel S, Powles SB. (2003b) An isoleucine residue within the carboxyl-transferase domain of multidomain acetyl-coenzyme A carboxylase is a major determinant of sensitivity to aryloxyphenoxypropionate but not to cyclohexanedione inhibitors. Plant Physiology 132:1716–1723.
Douce R, Joyard J, Block MA, Dorne AJ. (1990) Glycolipid analyses and synthesis in plastids. Methods in Plant Biochemistry 4:71–103.
Egli MA, Gengenbach BG, Gronwald JW, Somers DA, Wyse DL. (1993) Characterization of maize acetyl-CoA carboxylase. Plant Physiology 101:499–506.[Abstract]
Elborough KM, Winz R, Deka RK, Markham JE, White JA, Rawsthorne S, Slabas AR. (1996) Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenis. Biochemical Journal 315:103–112.
Emanuelsson O, Nielsen H, Brunak S, von Heijne G. (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. Journal of Molecular Biology 300:1005–1016.[CrossRef][ISI][Medline]
Giménez-Espinosa R, Plaisance KL, Plank DW, Gronwald JW, De Prado R. (1999) Propaquizafop absorption, translocation, metabolism, and effect on acetyl-CoA carboxylase isoforms in chickpea (Cicer arietinum L.). Pesticide Biochemistry and Physiology 65:140–150.[CrossRef]
Griffin JL and Habetz RJ. (1989) Soybean (Glycine max) tolerance to pre-emergence and post-emergence herbicides. Weed Technology 3:459–462.
Hart SE and Roskamp GK. (1998) Soybean (Glycine max) response to thifensulfuron and bentazon combinations. Weed Technology 12:179–184.
Hellgren LI and Sandelius AS. (2001) Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.). Journal of Experimental Botany 52:2275–2282.
Kapusta G, Jackson LA, Mason DS. (1986) Yield response of weed-free soybeans (Glycine max) to injury from postemergence broadleaf herbicides. Weed Science 34:304–307.
Kobek K, Focke M, Lichtenthaler HK. (1987) Fatty-acid biosynthesis and acetyl-CoA carboxylase as a target of diclofop, fenoxaprop and other aryloxy-phenoxy-propionic acid herbicides. Zeitschrift für Naturforschung 43:47–54.
Konishi T and Sasaki Y. (1994) Compartmentation of the two forms of acetyl-CoA carboxylase in plants and the origin of their tolerance toward herbicides. Proceedings of the National Academy of Sciences, USA 91:3598–3601.
Konishi T, Shinohara K, Yamada K, Sasaki Y. (1996) Acetyl-CoA carboxylase in higher plants: most plants other than Gramineae have both the prokaryotic and the eukaryotic form of this enzyme. Plant and Cell Physiology 37:117–122.
Kozaki A, Mayumi I, Sasaki Y. (2001) Thiol–disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase. Journal of Biological Chemistry 276:39919–39925.
Kozaki A and Sasaki Y. (1999) Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component. Biochemical Journal 339:541–546.
Lepage M. (1967) Identification and composition of turnip root lipids. Lipids 2:244–250.[Medline]
Luo XY, Sunohara Y, Matsumoto H. (2004) Fluazifop-butyl causes membrane peroxidation in the herbicide-susceptible broad leaf weed bristly starbur (Acanthospermum hispidum). Pesticide Biochemistry and Physiology 78:93–102.[CrossRef]
Madoka Y, Tomizawa K, Mizoi J, Nishida I, Nagano Y, Sasaki Y. (2002) Chloroplast transformation with modified accD operon increases acetyl-CoA carboxylase and causes extension of leaf longevity and increase in seed yield in tobacco. Plant and Cell Physiology 43:1518–1525.
Nelson KA and Renner KA. (2001) Soybean growth and development are affected by glyphosate and post-emergence herbicide tank mixtures. Agronomy Journal 93:428–434.
Nikolau BJ, Ohlrogge JB, Wurtele ES. (2003) Plant biotin containing carboxylases. Archives of Biochemistry and Biophysics 414:211–222.[CrossRef][ISI][Medline]
Nikolskaya T, Zagnitko O, Tevzadze G, Haselkorn R, Gorniki P. (1999) Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxytransferase domain. Proceedings of the National Academy of Sciences, USA 96:14647–14651.
Ohlrogge JB and Browse J. (1995) Lipid biosynthesis. The Plant Cell 7:957–970.[CrossRef][ISI][Medline]
Ohrogge JB and Jaworsky JG. (1997) Regulation of fatty acid synthesis. Annual Review of Plant Physiology and Plant Molecular Biology 48:109–136.[CrossRef][ISI]
Page RA, Okada S, Harwood JL. (1994) Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants. Biochimica et Biophysica Acta 1210:369–372.[Medline]
Rendina AR, Craig-Kennard AC, Beaudoin JC, Breen MK. (1990) Inhibition of acetyl-coenzyme A carboxylase by two classes of grass-selective herbicides. Journal of Agricultural and Food Chemistry 38:1282–1287.[CrossRef]
Reverdatto S, Beilinson V, Nielsen NC. (1999) A multisubunit acetyl-coenzyme A carboxylase from soybean. Plant Physiology 119:961–978.
Sasaki Y and Nagano Y. (2004) Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation and gene manipulation for plant breeding. Biosciences Biotechnology Biochemistry 68:1175–1184.
Sammons R, Pinter K, Widham JM. (1988) Effects of herbicide cyclohexanediones, a potent inhibitor of acetyl-CoA carboxylase from grass. Weed Technology 6:236–239.
Savage LJ and Ohlrogge JB. (1999) Phosphorylation of pea chloroplast acetyl-CoA carboxylase. The Plant Journal 18:521–527.[CrossRef][ISI][Medline]
Schulte W, Tôpper R, Stracke R, Schell J, Martini N. (1997) Multifunctional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform. Proceedings of the National Academy of Sciences, USA 94:3465–3470.
Shimabukuro RH, Davis DG, Hoffer BL. (2001) The effect of diclofop-methyl and its antagonist, vitamin E, on membrane lipids in oat (Avena sativa L.) and leafy spurge (Euphorbia esula L.). Pesticide Biochemistry and Physiology 69:13–26.[CrossRef]
Slabas AR, White A, O'Hara P, Fawcett T. (2002) Investigations into the regulation of lipid biosynthesis in Brassica napus using antisense down-regulation. Biochemical Society Transactions 30:1056–1059.[CrossRef][ISI][Medline]
Thelen JJ, Mekhedov S, Ohlrogge JB. (2001) Brassicaceae express multiple isoforms of biotin carboxyl carrier protein in a tissue-specific manner. Plant Physiology 125:2016–2028.
Towbin H, Staehelin T, Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76:4350–4354.
Walker KA, Ridley SM, Lewis T, Harwood JL. (1988) Fluazifop, a grass-selective herbicide which inhibits acetyl-CoA carboxylase in sensitive plant species. Biochemical Journal 254:307–310.[ISI][Medline]
Walker KA, Ridley SM, Lewis T, Harwood JL. (1989) Action of aryloxy-phenoxy carboxylic acids on lipid metabolism. Reviews in Weed Science 4:71–84.
Werck-Reichhart D, Hehn A, Didierjean L. (2000) Cytochrome P450 for engineering herbicide tolerance. Trends in Plant Science 5:116–123.[CrossRef][ISI][Medline]
Zagnitko O, Jelenska J, Tevzadze R, Gornicki P. (2001) An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors. Proceedings of the National Academy of Sciences, USA 98:6617–6622.
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