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Many plant phytochemicals constitute binary enzyme–glucoside systems and function in plant defence. In brassicas, the enzyme myrosinase is confined to specific myrosin cells that separate the enzyme from its substrate; the glucosinolates. The myrosinase-catalysed release of toxic and bioactive compounds such as isothiocyanates, upon activation or tissue damage, has been termed ‘the mustard oil bomb’ and characterized as a ‘toxic mine’ in plant defence. The removal of myrosin cells and the enzyme that triggers the release of phytochemicals have been investigated by genetically modifying Brassica napus plants to remove myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a ribonuclease, barnase. Transgenic plants ectopically expressing barnase were embryo lethal. Co-expressing barnase under the control of the Myr1.Bn1 promoter with the barnase inhibitor, barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of myrosinase and negligible myrosinase activity, and autolysis experiments showing negligible production of glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named ‘MINELESS plants’. The epithiospecifier protein profile and glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35kDa epithiospecifier protein in myrosin cells and a reduced turnover of glucosinolates in MINELESS plants.
Glucosinolates (GSLs) are a group of allelochemicals that are present in the order Capparales, containing the Brassicaceae family (Sørensen, 1990; Bellostas et al., 2007). GSLs are hydrolysed by myrosinase (MYR) upon plant tissue rupture and used as defence chemicals against herbivores. GSLs are nitrogen- and sulphur-containing secondary metabolites that share a core consisting of a β-thioglucoside moiety and a sulphonated oxime, but differ by a variable side chain derived from one of several amino acids (Kliebenstein et al., 2001; Mithen, 2001a; Grubb and Abel, 2006; Tripathi and Mishra, 2007; Yan and Chen, 2007).
The enzyme MYR (EC 18.104.22.168) is a β-thioglucosidase that hydrolyses GSLs to a variety of products such as isothiocyanates, thiocyanates, nitriles, epithionitriles, and oxazolidine-thiones depending on the nature of the GSL, reaction conditions, and the presence of protein cofactors (Bones and Rossiter, 1996, 2006; Rask et al., 2000). These protein cofactors include epithiospecifier protein (ESP), nitrile-specifier proteins (NSPs), and thiocyanate-forming protein (TFP) which in turn depend on ferrous/ferric ions (Bones and Rossiter, 1996; Foo et al., 2000; Lambrix et al., 2001; Burow et al., 2007a; Kissen and Bones, 2009).
MYRs are present in specialized cells known as ‘myrosin cells’ (Bones and Iversen, 1985; Thangstad et al., 2004; Kissen et al., 2009) that are dispersed throughout plant tissues. MYR is present in palisade parenchyma, phloem idioblasts, guard cells, and ground tissue cells (Bones and Iversen, 1985; Bones et al., 1991; Andreasson et al., 2001). Immunocytochemical and in situ hybridization studies carried out on seeds of Brassicaceae have shown MYR to be exclusively present in myrosin cells of embryonic cotyledons and the radicle periphery (Thangstad et al., 1990, 1991; Bones et al., 1991; Höglund et al., 1992; Geshi and Brandt, 1998; Kelly et al., 1998; Husebye et al., 2002; Kissen et al., 2009).
The localization of GSLs is still unclear, although some studies have shown sinigrin to be localized in all cells except myrosin cells of Brassica juncea seeds (Kelly et al., 1998), and radiolabelled GSLs appear to be transported to specific cells (Thangstad et al., 2001). In Arabidopsis thaliana flower stalks, GSLs are thought to be present in S-cells (sulphur-rich cells) (Koroleva et al., 2000) and are neighbours to the scattered idioblast cells containing MYR, which have been named myrosin phloem cells (Husebye et al., 2002). The MYR genes in Brassica napus can be divided into three subfamilies, MA, MB, and MC (Xue et al., 1992; Falk et al., 1995). There are in total ~25 MYR-encoding genes in B. napus, two of which have been characterized and cloned, and which are members of MYR subfamilies MA (Myr1.Bn1) and MB (Myr2.Bn1), respectively (Lenman et al., 1993; Thangstad et al., 1993). Myr1.Bn1 is a myrosin cell-specific gene which displays a highly specific expression in seed myrosin cells. The expression from its promoter has been shown to be restricted to this cell type (Thangstad et al., 2004).
The ESP has been described as a MYR cofactor that drives the hydrolysis of alkenyl GSLs such as progoitrin, sinigrin, and gluconapin towards the production of epithionitriles (cyanoepithioalkanes) and alkyl GSLs towards nitriles instead of other possible hydrolysis products (MacLeod and Rossiter, 1985; Bernardi et al., 2000; Foo et al., 2000; Lambrix et al., 2001; Kissen and Bones, 2009). Ferrous/ferric ions and MYR together with the ESP are essential for epithionitrile formation (Macleod and Rossiter, 1987; Zabala et al., 2005; Burow et al., 2006). In B. napus, two isoforms of ESP polypeptides with molecular masses of 39kDa and 35kDa have been purified, partially sequenced, and characterized (Bernardi et al., 2000; Foo et al., 2000). Two anti-ESP antibody-reactive bands of 37kDa and 43kDa have also been observed in broccoli and cabbage (Matusheski et al., 2006).
Oilseed rape meal is a high protein feed for livestock. It contains the GSLs progoitrin or epiprogoitrin, gluconapin, and glucobrassicanapin, and has mostly been used in ruminants. Intact GSLs can serve as contact cues for feeding or oviposition stimulation (Halkier and Gershenzon, 2006). Moreover, MYR activity is also considered to be important for plant defence against specialist insects that have adapted themselves to intact GSLs, but less important for defence against generalists, which are susceptible to intact GSLs (Li et al., 2000).
GSLs are themselves biologically inactive, but GSL hydrolytic products (thiocyanates, isothiocyanates, nitriles, and oxazolidine-2-thione) produced by the enzyme MYR during processing of oilseed rape meal are biologically active (Mawson et al., 1993). The thiocyanates interfere with iodine availability, whereas 5-vinyl-1,3-oxazolidine-2-thione is responsible for the morphological and physiological changes of the thyroid and leads to goitre formation. Nitriles are known to affect liver and kidney functions (Elfving, 1980; Mithen, 2001b; Bellostas et al., 2007; Tripathi and Mishra, 2007). Thus it is important to devise technologies that prevent the hydrolysis of GSLs in ruminants as an alternative to lowering the concentration of GSLs.
The MYR-catalysed release of toxic and bioactive compounds such as isothiocyanates by hydrolysing GSLs, upon activation or tissue damage, has been termed ‘the mustard oil bomb’ (Matile, 1980; Bones and Rossiter, 1996). The biological/ecological significance of the GSL–MYR system has been the subject of interest for several years, has been analysed to a large extent, and reviewed (Rask et al., 2000; Kliebenstein et al., 2005; Hopkins et al., 2008; Müller, 2009; Textor and Gershenzon, 2009). An ecological role for ‘the mustard oil bomb’ from seeds has been studied by analysing the GSL–MYR defence system in B. juncea cotyledons during seedling development in defence against the generalist herbivore, Spodoptera eridania (Wallace and Eigenbrode, 2002), by testing the seed nutritional quality against the yellow meal worm/common beetle generalist (Tenebrio molitor) (Davis and Sosulski, 1974; Davis et al., 1981, 1983; Eriksson et al., 2002), and as an allelochemical in Brassica nigra (Lankau and Strauss, 2007).
The objective of this study was to produce transgenic B. napus plants with seeds that lack myrosin cells. Ablation of cells and tissue by the controlled expression of lethal genes has been performed previously, but its widespread success has often been limited by secondary effects on non-targeted tissue. Genetic ablation studies in plants have focused on engineering of male and female sterility, blocking anther dehiscence and sexual reproduction in, for example, tobacco, tomato, wheat, and populous trees, and genetic ablation of flowers in A. thaliana (Goldman et al., 1994; Beals and Goldberg, 1997; De Block et al., 1997; Nilsson et al., 1998; Goetz et al., 2001; Roque et al., 2007; Wei et al., 2007; Wang et al., 2008). The objective of this study was to produce transgenic B. napus plants with seeds that lack myrosin cells using a genetic ablation strategy. The very first genetic cell ablation strategy induced male sterility in B. napus with the barnase gene regulated by the tapetum-specific TA 29 promoter (Mariani et al., 1990). Barnase is a 110 amino acid extracellular potent RNase that is secreted by the bacterium Bacillus amyloliquefaciens and that is used as a digestive enzyme for nutritional purposes or/and as a defence toxin. Barstar is an 89 amino acid intracellular inhibitor of barnase that is produced constitutively by the bacterium. Barstar binds specifically to barnase, forming inactive barnase–barstar complexes (Hartley, 1989).
In the present study, the Myr1.Bn1 gene promoter was used for this purpose, because Myr1.Bn1 expression has been shown to be restricted to myrosin cells (Thangstad et al., 2004). The expression of cytotoxic barnase driven by the Myr1.Bn1 gene promoter resulted in controlled cell death of myrosin cell idioblasts. Not unexpectedly, the expression of barnase only (pMyr1.Bn1:Barnase) was embryo lethal. Due to the very toxic nature of barnase, barstar has often been co-expressed under the control of a constitutive promoter, to avoid plant lethality. It has also been shown that the co-expression of barstar under the control of a constitutive cauliflower mosaic virus (CaMV) promoter 35S leads to the ablation of myrosin cells without secondary effects in seeds. Controlled cell death (ablation) of myrosin cells produced MINELESS seeds—seeds with a dramatic reduction of MYR-containing toxic mines. The genetic ablation was successfully achieved using the promoter constructs pMyr1:Barnase in combination with 35S:Barstar.
The sequence of the Myr1.Bn1 gene is given in GenBank (accession Z21977.3). The cloning procedure of the Myr1.Bn1 promoter is as described by Thangstad et al. (2004). Standard molecular biology methods were employed (Sambrook et al., 1989). Escherichia coli DH5α (Bethesda Research Laboratories), JM109 (Promega, Madison, WI, USA), and MX1061 (Plant Genetic Systems, Ghent, Belgium) were used for plasmid manipulations. Because of the toxicity of barnase, all plasmids containing this gene were propagated in the E. coli MX1061 strain, which has a chromosomal expression of the barnase inhibitor gene barstar. Plasmids pBluescript II KS (Stratagene, La Jolla, CA, USA) and pGEM3, 5, and 11 (Promega) were used for subcloning. Briefly, the procedure for cloning is as follows. A SalI–EcoRI 1142bp fragment containing the partial Myr1.Bn1 promoter, the barnase-encoding gene (Mariani et al., 1990), and the Nos terminator (Depicker et al., 1982) was constructed in several cloning steps and inserted into pBI101.1, between the SalI and EcoRI restriction sites of the polylinker. The resulting plasmid was cut with HindIII–SalI and the remaining 2520bp of the Myr1.Bn1 promoter inserted utilizing the internal SalI site to obtain a binary plasmid carrying the full-length promoter, linked to the barnase gene and a Nos terminator, the pMyr1.Bn1:Barnase construct (Fig. 1A). To generate the pMyr1.Bn1:Barnase:Barstar plasmid construct (MyrBarnBar=MINELESS), a cassette consisting of the barstar gene (Mariani et al., 1992) under the CaMV35S promoter with a 3′g7 terminator (Velten and Schell, 1985) was inserted at the EcoRI restriction site of pBI101.1 containing the 1142bp fragment described above and the 2520bp pMyr1.Bn1 promoter fragment inserted, giving rise to a plasmid containing the full-length MYR Myr1.Bn1 promoter, barnase, Nos terminator, and CaMV35S:Barstar:3′g7 terminator (Fig. 1B). The constructs shown were verified by restriction digests and sequencing. The two constructs were transformed into Agrobacterium tumefaciens strain LBA4404 (Clontech, Palo Alto, CA, USA) by electroporation and used to transform B. napus.
Transformation of B. napus was performed essentially as described by Moloney et al. (1989). Seeds of B. napus cv. Westar were surface-sterilized in 1% sodium hypochlorite for 20min, washed in sterile water three times, and planted in jars containing MS medium (pH 5.8) (Murashige and Skoog, 1962) supplemented with 1% sucrose and 0.8% agar gel (Sigma). Seeds were then germinated under controlled conditions at 22°C in a 16h light/8h dark photoperiod and at a light intensity of 70–80μmol m−2 s−1. The cotyledons with the cotyledonary petioles from 5-day-old seedlings were used as the explants for transformation using A. tumefaciens LBA4404 containing the binary plasmid constructs pMyr1.Bn1:Barnase and pMyr1.Bn1:Barnase:Barstar. After co-cultivation, the selection of transformants was done on the basis of kanamycin resistance from the NPTII gene in the constructs. After regeneration of transgenic shoots, the verification of inserted constructs was conducted using PCR with genomic DNA as the template and specific primers for the inserted genes (barnase and barstar) essentially as described by Strittmatter et al. (1995). After repetitive transfers and hormone-induced rooting, plants (designated T0) were transferred to soil and grown in controlled-environment rooms. Plants were allowed to self-pollinate and flowers were covered with paper bags. At maturity, the siliques were harvested and seeds (designated T1) were collected from three transgenic lines of the pMyr1.Bn1:Barnase:Barstar construct and were verified by MYR activity and quantitative real-time PCR (qRT-PCR). Only one line from the pMyr1.Bn1:Barnase construct was obtained, and due to embryo lethality seeds could not be further characterized.
The MYR activity was measured using the GOD-Perid assay (Bones and Slupphaug, 1989), by measuring the amount of glucose liberated from hydrolysis of the GSL sinigrin. The assay was performed using citrate buffer (50mM, pH 5.5), sinigrin (15mg ml−1), and GOD-Perid reagent (Roche, Basel, Switzerland). In order to calculate specific MYR activity, the total protein content of samples was also measured using Bradford reagent (BioRad Laboratories, UK). The specific MYR activity is described as nmol glucose min−1 mg−1 protein.
QRT-PCR was performed to verify the expression of the transgenes in the three selected lines of MINELESS (pMyr1.Bn1:Barnase:Barstar). Frozen cotyledons from the wild type (Westar) and transgenic (MINELESS) lines were homogenized in pre-cooled (–80°C) 2.0ml tubes containing 5mm stainless steel beads for 2min at full speed using a Tissuelyser homogenization device (Qiagen, Valencia, CA, USA). Total RNA was extracted with a Spectrum Plant Total RNA Kit (Sigma-Aldrich), additionally treated with on-column DNase (RNase-free DNase Set, Qiagen), and RNasin® ribonuclease inhibitor (1U μl−1) (Promega Corporation, Madison, WI, USA) was added. The RNA was quantified using NanoDrop ND 1000 (Nanodrop Technologies, Wilmington, DE, USA) and analysed by formaldehyde–agarose gel electrophoresis. The cDNA was synthesized from 1μg of total RNA using the QuantiTect® Reverse Transcription Kit (Qiagen). The sequences for gene-specific primers used in qRT-PCR are given in Supplementary Table S1 avaiable at JXB online. The qRT-PCR was carried out using the LightCycler 480 SYBR Green I Master Kit (Roche, Basel, Switzerland) following the manufacturer's instructions. Each 20μl reaction contained 0.5μM of each forward and reverse primer, and a cDNA quantity corresponding to 0.05μg of total RNA. PCR was performed in a LightCycler 480 (Roche, Basel, Switzerland) as follows: (i) 5min at 95°C; (ii) 45 amplification cycles; 95°C for 10s, 55°C for 15s (Barstar and ESP), or 53°C for 15s (MYR); 72°C for 11s; and (iii) 95°C for 5s, 65°C for 1min and 97°C continuously for analysis of dissociation curves. Cp values and melting curves were calculated by the LightCycler 480 analysis programs using the second derivative maximum method. The PCR efficiency was determined employing LinReg PCR (Ramakers et al., 2003) and the relative expression ratios were calculated by using the Relative Expression Software Tool (REST) 2005 V.1.9.12) (Pfaffl et al., 2002). The RNA amount per sample was normalized using Allene oxide synthase as a reference gene.
To proceed with the structural analysis of myrosin cells through light, fluorescence, and transmission electron microscopy and GSL quantification, wild type and MINELESS seeds were plated on moistened filter paper-lined Petri dishes and were kept imbibed for 4h at room temperature. To select MINELESS seeds with low or negligible MYR activity, a fast version of the GOD-Perid assay was performed as follows. After removal of seed coats from imbibed seeds, one cotyledon from a single seed was crushed with a solution of citrate buffer (50mM, pH 5.5) and sinigrin (15mg ml−1), GOD-Perid (750μl) was then added and after an incubation of 5min the development of green colour indicated MYR activity. The other cotyledons from MINELESS single seeds (i.e. seeds that gave no green colour/very light green colour in the GOD-Perid assay) were further processed for structural analysis of myrosin cells as described below. In parallel, Westar wild type seeds were also run to see the colour differences between the wild type and MINELESS seeds.
Cotyledons of imbibed seeds of MINELESS seeds and wild type seeds were fixed, dehydrated, and embedded as described by Thangstad et al. (1991) with 6d of infiltration with LR-White. This material was further sectioned into 1μm and 600–700Å thick sections to observe myrosin cells under a light microscope and a transmission electron microscope. For light microscopy, semi-thin sections were stained with toluidine blue. Slides were examined and photographed with a research microscope (Eclipse 800; Nikon, Tokyo, Japan), equipped with a digital camera (SPOT RT; Diagnostic Instruments, Burroughs, MI, USA). Semi-thin sections were used for the detection of MYR with the polyclonal antibody K089 (Thangstad et al., 1991). Positive cells were visualized with fluorescein isothiocyanate (FITC)-conjugated streptavidin (DAKO, Glostrup, Denmark). The immunogold labelling of thin sections was carried out as described in Thangstad et al. (1991) with minor modifications. The goat anti-rabbit secondary antibody (Amersham, Buckinghamshire, UK) was conjugated with 15nm colloidal gold, and the post-embedding on-grid osmium staining was performed with 2% osmium tetroxide. The sections were examined and micrographs were taken with a Jeol 1200 EX (Japan) electron microscope at 60kV.
Single seeds and 5-day-old cotyledons of wild type and transgenic (MINELESS) lines were extracted in 100μl of imidazole-HCl buffer (10mM, pH 6.0). Single seeds were defatted before extraction with n-hexane and the pellet was air dried. The samples were centrifuged at 4°C, and supernatants were transferred to dialysis membranes (12000–14000Da MWCO) (Spectra/Por), dialysed overnight in imidazole-HCl buffer, centrifuged at 4°C, and used for MYR activity assay and protein assays.
For the analysis of expression of MBPs, another set of single seeds was ground in liquid N2, transferred to 100μl of imidazole-HCl buffer (10mM, pH 6.0) containing phenylmethylsulphonyl fluoride (PMSF; 1mM), and incubated for 30min at 4°C before centrifugation and sampling of the supernatant.
The MYR and MBP extracts were further processed for SDS–PAGE and immunoblot analysis.
A 10μg aliquot of protein samples was solubilized in standard SDS buffer prior to separation by SDS–PAGE using 7.5% and 12% polyacrylamide gels in a Hoefer miniVe vertical electrophoresis system. For immunoblotting, gels were blotted onto a 0.45μm nitrocellulose membrane (Bio-Rad) in Hoefers blot module as described by the manufacturer. MYRs and MBPs were detected by the 3D7 and 34:14 monoclonal antibodies, respectively (Lenman et al., 1990). The detection was visualized using a chemiluminescent detecting system (Super Signal West Pico, Pierce, Rockford, IL, USA).
Wild type and MINELESS single seeds were extracted in 0.6ml of imidazole-HCl buffer (100mM, pH 6.8). A part of the extract from single seeds was used for determination of MYR activity by GOD-Perid assay, separation by SDS–PAGE, and immunoblot analysis. Immunoblot analysis was carried out as described above, and the ESP was detected by the anti-ESP polyclonal antibody characterized earlier (Foo et al., 2000). The rest of the extract was used for ESP activity assay. To proceed with the ESP activity assay, the extract was subdivided into two parts. One part was incubated in an assay mixture containing ferrous ions (0.6mM ferrous ammonium sulphate), dithiothreitol (DTT; 1mM), sinigrin (14mM), and MYR (50μl from an affinity-purified preparation from Sinapis alba seed with the ability to hydrolyse 865μmol sinigrin min−1 as tested by GOD-Perid), while in the other part MYR was omitted. 1-Dodecanol was used as an internal standard. After 4h incubation at 30°C, the mixtures were extracted twice with dichloromethane and the combined organic phases dried with anhydrous MgSO4, and concentrated using a flow of nitrogen gas. ESP activity was determined by GC-MS on a Varian Star 3400 CX gas chromatograph coupled with a Varian Saturn 3 mass spectrometer. A polar column, Chrompack CP-Wax 52CB (30m×0.32 id×0.25μm film thickness), was used for all separations. The carrier gas flow (He) was held at 50ml min−1 (injector) and 30 cms−1 (column). The injector was kept at 200°C (split injection), and the GC temperature program was held at 35°C for 1min, followed by 40–210°C at a rate of 7°C min−1. The MS detector was set at 200°C, and a mass range of m/z 40–300 was recorded (EI mode at 70eV). GSL hydrolysis products were tentatively identified by using a mass spectra database search (NIST MS Database, 1998), and by comparing with mass spectra from the literature (Spencer and Daxenbichler, 1980).
Wild type and MINELESS seeds were cut into two parts, with one part used for GSL analysis and one part for MYR activity assay. After recording the dry weight, half of the seed was extracted with 3× 1ml methanol (70%) at 70°C. The extracts were heated at 70°C in a heated block (Techne, Dry-Block, DB-2P) for 10min. After cooling, the samples were centrifuged at 15000g for 10min at 4°C. The supernatants, stored on ice between the three extractions, were then combined and filtered through 0.45μm filters (Millipore Corporation, Bedford, MA, USA). GSLs were desulphonated on a DEAE-A25 Sephadex ion exchange column (Amersham Biosciences, Uppsala, Sweden) supplemented with sulphatase (50μl, Type H-1, from Helix pomatia, Sigma, St Louis, MO, USA) that was prepared as described by Graser et al. (2001). Benzyl GSL was used as an internal standard. The desulpho-GSLs were freeze-dried (Virtis Benchtop), reconstituted in water (100μl), and analysed by HPLC (Agilent HP 1100 Series). Reverse-phase HPLC was performed on a Supelcosil LC 18 (250mm×2.1mm, 5μm spherical particles) Supelco column (Bellefonte PA, USA). The mobile phases were: (A) deionized water and (B) acetonitrile. The following gradient was used: 0–2min, 3% B; 2–17min, 3–40% B; 17–22min, 40% B; 22–22.10min, 40–100% B; 22.10–32min, 100% B; 32–32.10min, 100–3% B; 32.10–50min, 3% B. The flow rate was 0.3ml min−1 with UV detection at 229nm. The injection volume was 50μl. The LC-MS analysis was performed using a HPLC-DAD (Agilent HP 1100 Series) coupled to an Agilent 1100 Series LC/MSD trap mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with an APCI interface. The instrument was configured in positive ion chemical ionization. The column, solvents with gradient, flow rate, and injection volume were the same as described above for HPLC. The APCI settings were: nebulizer pressure, 60psi; drying gas flow, 51 min−1; drying gas temperature, 350°C; APCI vap temperature, 400°C; corona current, 4000nA; capillary voltage, 3500V; compound stability; 100%; trap drive level, 100%. The individual GSLs were identified by diode array UV spectra and mass spectra. The identity of the compounds was confirmed by comparison with retention times, UV spectra, and MS spectra of authentic standard desulpho-GSLs (mixture of isolated B. napus desulpho-GSLs, BIORAF foundation, Denmark). The correction factors at 229nm from Brown et al. (2003) and Daun and McGregor (1991) were used to calculate the concentration of the GSLs.
Three out of four kanamycin-resistant T0 lines of pMyr1.Bn1:Barnase (Fig. 1A) amplified the expected 160bp PCR product from the inserted barnase gene (Strittmatter et al., 1995), and were transferred to soil. Only one of these plants developed to maturity. Three out of six kanamycin-resistant T0 lines of pMyr1.Bn1:Barnase:Barstar (Fig. 1B) were verified positive, amplified the expected 160bp product from barnase and the 235bp product from barstar (Strittmatter et al., 1995; data not shown), and were transferred to soil.
The morphological appearance of pMyr1.Bn1:Barnase and pMyr1.Bn1:Barnase:Barstar transgenic (T0) lines showed major differences. The single transgenic pMyr1.Bn1:Barnase line obtained after transfer to soil produced a viable plant, but no viable seeds. The majority of the embryos were terminated during silique development, and showed necrosis and altered morphology. Due to this embryo lethality, viable seeds could not be further characterized. Transgenic lines with the genetic construct pMyr1.Bn1:Barnase:Barstar had a close to normal morphological phenotype, where flowering was unaffected and viable seeds were produced.
To confirm the production of transgenic B. napus plants, 5-day-old cotyledons of three different transgenic T1 lines of pMyr1.Bn1:Barnase:Barstar were analysed for barstar expression. All three transgenic lines showed barstar expression, while no barstar expression was detected in wild type Westar as verified by qRT-PCR (data not shown).
Seeds from three transgenic T1 lines of pMyr1.Bn1:Barnase:Barstar were analysed for MYR activity by enzymatic glucose release using sinigrin as a substrate. All tested seeds (n=7–8) of transgenic lines TL1 and TL2, and five out of seven seeds of TL3, had lower MYR activity than the lowest MYR activity value of the wild type. Moreover, 5-day-old cotyledons of three transgenic lines and the wild type showed significantly low MYR activity (nmol glucose min−1 mg−1 protein) in all three transgenic lines as compared with wild type Westar (Supplementary Fig. S1B at JXB online).
The gene expression of GSL metabolism genes Myr1 and ESP by qRT-PCR showed significant down-regulation in 5-day-old cotyledons of three transgenic lines as compared with the wild type (Supplementary Fig. S2 at JXB online). The Myr1 and ESP genes showed down-regulation with log2 ratios of –3.8, –5.3, and –2.4, and–3.4, –4.1, and –2.6, respectively, in three transgenic lines (TL1, TL2, and TL3) (P-values: Myr1 <0.01, 0.00, 0.05; and ESP <0.05, 0.01, 0.00).
The transgenic line (TL1) that showed low MYR activity for both seeds (30.4nmol glucose min−1 mg−1 protein) and cotyledons (7.9nmol glucose min−1 mg−1 protein) was selected for further characterization. These seeds were termed MINELESS, as the ‘toxic mines’ (myrosin cells) had been genetically ablated in the mature seeds.
The targeted myrosin cells from semi-thin sections of MINELESS seeds (radicles and cotyledons) stained with toluidine blue appeared empty when observed under light microscopy (Fig. 2A, B) while wild type tissues showed the expected distribution of myrosin cells (Bones et al., 1991). A myrosin cell from a wild type section stained with toluidine blue is shown as an insert in Fig. 2B. Semi-thin sections (1μm) were prepared for immunofluorescence microscopy. The anti-MYR antibody K089 (Thangstad et al., 1991) followed by FITC-conjugated secondary antibody provided specific labelling for myrosin cells in wild type sections (Fig. 2C) while no specific labelling could be seen in MYR-negative MINELESS seed sections (Fig. 2E). MINELESS seeds with a weak/moderate MYR activity (described later) (Fig. 4) had both empty myrosin cells and myrosin cells with partially intact myrosin grains (Fig. 2D). It was also possible to observe different stages of cell degradation, with both unablated and semi-ablated myrosin cells.
The myrosin cell structures from wild type and MINELESS seeds were further analysed using transmission electron microscopy and immunogold-electron microscopy (EM). Myrosin cells of the wild type had a typical even granulated matrix in the myrosin grains (protein bodies/protein vacuoles) (Fig. 3A). In MINELESS plants, these cells appeared electron transparent and shrunken (Fig. 3B, C), proving that the cell content had been ablated. The surrounding aleurone-like cells appeared structurally normal, showing that the achieved ablation was highly cell specific. Further ultrastructural analysis using immunogold-EM of thin sections (600–700Å) and the anti-MYR antibody K089 (Thangstad et al., 1991) showed that the ablated cells, or other surrounding tissue, did not contain MYR (Fig. 3E). While protein bodies/vacuoles of myrosin cells from wild type tissues were densely labelled with immunogold particles localizing MYR (Fig. 3D and insert), no labelling was seen in MINELESS tissues (Fig. 3E and insert). It could also be observed that myrosin cells activated for ablation had characteristics of apoptosis such as nuclear membrane blebbing (Fig. 3B). A notable feature of the ablated cells is the apparent absence of any effect on neighbouring cells (Fig. 3E and insert).
In total 100 MINELESS and 50 Westar wild type single seeds were tested for MYR activity using sinigrin as a substrate. The distribution of specific MYR activity of wild type and transgenic single seeds is shown in Fig 4. Sixty percent of wild type seeds possessed MYR activity in the region of 200–400nmol glucose min−1 mg−1 protein, while almost all (96%) transgenic seeds were found to have activity <200 nmol glucose min−1 mg−1 protein. Seeds with no or negligible activities (i.e. <10 nmol glucose min−1 mg−1 protein) were classified as MINELESS seeds. Of the transgenic seeds examined, 23% were found in this category (Fig. 4). Out of 50 wild type seeds that were tested for MYR activity, most seeds showed MYR activity in the range of 201–400nmol glucose min−1 mg−1 protein and only a few seeds had MYR activity beyond this range (Fig. 4).
Immunoblot analysis using an anti-MYR monoclonal antibody (3D7 antibody) specifically labelled the three major bands for MYR polypeptide classes of B. napus, denoted as 75, 70, and 65kDa in wild type protein extracts (Fig. 5A). All these three bands were absent in the protein extracts from MINELESS seeds.
Through immunoblot analysis and by using the monoclonal anti-MBP antibody 34:14 (Lenman et al., 1990) that recognizes different MBPs (Falk et al., 1995; Geshi and Brandt, 1998; Eriksson et al., 2002), no clear differences were observed between the wild type and MINELESS seeds (Fig. 5B). In some seeds, including both wild type and MINELESS, the 110kDa MBP isoform was only faintly expressed, while in others it was strongly expressed. The 52kDa MBP band was visible in all MINELESS seeds examined, but the band intensity was low compared with that in wild type seeds.
The effect of myrosin cell ablation on GSL hydrolysis and ESP activity was determined on single MINELESS and wild type seeds. GC-MS analysis was carried out on individual autolysed seeds using sinigrin as a substrate. From wild type seeds the expected allyl isothiocyanate (All ITC), 3,4-epithiobutane nitrile (3,4ETBut NIT), and allyl nitrile (All NIT) were produced upon hydrolysis of the GSL sinigrin due to the presence of endogenous MYR and ESP (Fig. 6A). MINELESS seeds showed a greatly reduced ability to hydrolyse the GSL sinigrin (Fig. 6B). In MINELESS seeds, the total hydrolytic product formation was <3.3 nmol hydrolysis product mg−1 seed as compared with 39.8 nmol hydrolysis product mg−1 seed for the wild type (Fig. 6C). These differences in the production of hydrolytic products between the wild type and MINELESS were significant (P <0.02).
Addition of MYR purified from S. alba (Bones and Slupphaug, 1989) restored the GSL hydrolysis capacity in MINELESS extracts (Fig. 6C). Immunoblot analysis using a polyclonal anti-ESP antibody (Foo et al., 2000) detected two bands of 35kDa and 39kDa in wild type seeds (Fig. 6D). In comparison, MINELESS seeds lacked the 35kDa isoform (Fig. 6D). As expected from the low ESP levels detected in the immunoblot experiments, the production of allyl nitrile and 3,4-epithiobutane nitrile was much lower in MINELESS than in wild type extracts, which presumably are due to low MYR in MINELESS extracts (Fig. 6B). After addition of exogenous MYR, the only significant difference was the lower production of 3,4-epithiobutane nitrile in MINELESS as compared with the wild type (P <0.01) (Fig. 6C).
The total GSL content and corresponding GSL profile was analysed from seeds split into two equal halves; the remainder was used for analysis of MYR activity. MINELESS seeds which showed MYR activity in the range of 0–10nmol glucose min−1 mg−1 protein (Fig. 7A) were analysed for GSL. The total GSL concentration was increased in MINELESS seeds as compared with the wild type (Fig. 7B) (P <0.01). The aliphatic GSLs in MINELESS seeds were >2-fold higher than in wild type seeds. 2(R)-2-hydroxy-3-butenyl (progoitrin), 5-methylsulphinylpentyl (glucoalyssin), 2-hydroxy-4-pentenyl (gluconapoleiferin), 4-pentenyl (glucobrassicanapin), and 4-hydroxy-3-indolylmethyl (4-hydroxyglucobrassicin) were increased in MINELESS seeds as compared with the wild type, with progoitrin being the highest (Fig. 7C). Aliphatic GSLs in MINELESS seeds were >2-fold higher than in the wild type seeds. All aliphatic GSLs except 3-butenyl (gluconapin), and the indolic GSL 3-indolylmethyl (glucobrassicin) showed a significant increase in MINELESS seeds (P < 0.04).
The distribution of MYR activity versus total GSL content from each examined MINELESS seed is visualized in a scatter plot (Fig. 8). The total GSL content ranged from 6nmol GSL mg−1 seed to 37nmol GSL mg−1 seed. The majority of MINELESS seeds (10 out of 15 seeds), however, contained 10–30nmol GSL mg−1 seed (Fig. 8). In contrast, most of the wild type seeds possessed a total GSL content in the range of 10–13nmol GSL mg−1 seed, and two wild type seeds were even below this range (Fig. 8).
The detection of barstar only in transgenic lines and lack of its expression in the wild type confirmed the production of transgenic MINELESS plants. Moreover, the significantly reduced MYR activity in both seeds and cotyledons and strong down-regulation of glucosinolate metabolism genes (Myr1 and ESP) in three transgenic lines as compared with the wild type by qRT-PCR analysis provided confirmation of the production of transgenic MINELESS B. napus plants (Supplementary Figs S1, S2 at JXB online). Myr1 and ESP are the important genes in the GSL degradation pathway, and the strong down-regulation of these genes in transgenic lines confirms removal of myrosin cells. An almost similar level of down-regulation of the transcripts of Myr1 and ESP as measured with qRT-PCR provides another confirmation that ESP is associated with MYR. ESP has previously been described as a MYR cofactor and plays an important role during GSL degradation (MacLeod and Rossiter, 1985; Bones and Rossiter, 1996; Bernardi et al., 2000; Foo et al., 2000; Burow et al., 2009; Kissen and Bones, 2009).
The use of T-DNA insertion knockouts in A. thaliana has made possible a large-scale elucidation of biochemical signalling pathways. However, this approach has limitations in the case of redundancy of members within gene families, as found in the MYR genes and its substrate pathways (Barth and Jander, 2006). An alternative cell-specific strategy to remove MYR from B. napus seed was therefore embarked on.
Since MYR is present in myrosin cells, transgenic seeds were expected to be free of MYR activity. This would prevent GSL hydrolysis and formation of anti-nutritional compounds in seed meal fed to ruminants. The term MINELESS seeds has been adopted to illustrate that the ‘the toxic mines’ (myrosin cells) have been removed. The present genetic cell ablation study has proven that the expression of a cell-specific cytotoxic construct encoded by the RNase barnase, driven by the stringent cell-specific Myr1.Bn1 promoter, results in controlled cell death of myrosin cells in MINELESS seeds.
Previously, genetic ablation has been used as a tool to study male sterility. Tapetum-specific promoters were fused to cytotoxic genes and were shown to cause controlled cell death during the development phase (Nasrallah et al., 1991; Block and Debrouwer, 1993; Roberts et al., 1995; Zhan et al., 1996; Beals and Goldberg, 1997; De Block et al., 1997; Lee et al., 2003; Roque et al., 2007). The use of barnase has been limited by the ectopic expression and disruption of non-targeted tissue. The usefulness of barnase as a factor leading to cell ablation apparently depends on the specificity of the promoter used to control its expression. In the current study, a MYR promoter was used to direct the expression of cytotoxic barnase specifically to myrosin cells (pMyr1:Barnase). As a result, the barnase gene was strongly expressed in myrosin cells of maturing embryos/developing seeds of transgenic plants. However, due to the high toxicity of barnase, the viable seeds could be obtained only in the case of the co-expression of the barnase inhibitor barstar [pMyr1.Bn1:Barnase:Barstar (MyrBarnBar=MINELESS)]. Formation of inactive complexes (barnase–barstar) protects the embryo from widespread cell death, and restricts the ablation to single myrosin cells. The KD of a barnase–barstar complex has been estimated to be ~10−15M. A KD in this range indicates that once barnase–barstar complexes are formed, they are highly stable and rarely dissociate (Goldberg et al., 1995). The fact that MINELESS seeds could germinate normally confirms the relative stability of the barnase–barstar complex.
The empty holes, visualizing the ablated myrosin cells, had the same distribution as myrosin cells in wild type Brassicaceae (Bones and Iversen, 1985; Bones et al., 1991; Thangstad et al., 1991; Höglund et al., 1992). Structural analysis did not provide any indications suggesting that other cells were affected by the myrosin cell ablation. The use of transmission electron microscopy made it possible to examine the ultrastructure of the targeted myrosin cells in MINELESS in comparison with the wild type. Different levels of ablation were observed, from partial to completely empty remains surrounded by a cell wall (Fig. 3B, C, E). The identity of the ablated cells as myrosin cells was confirmed by immunogold-EM labelling of myrosin grains in wild type myrosin cells (Fig. 3D) compared with the absence of labelling in MINELESS seeds (Fig. 3E).
The MYR activity in transgenic seeds was reduced, but varied considerably among single seeds (Fig. 4). This was expected due to segregation of the transgenes and possibly also due to the cell- and time-specific variation of the pMyr1Bn1 promoter. MYR activity and GSL levels in the wild type are controlled by several genes and can be regarded as a continuous trait in Arabidopsis (Pfalz et al., 2007), and is even more complex in B. napus. The continuous trait variation is also apparent in MINELESS, as is visible in Fig. 4, indicating a segregation of the T1 MINELESS phenotype. Despite the observed variability, most of the transgenic seeds showed MYR activity <40nmol glucose min−1 mg−1 protein. The major fraction of measured MYR activities from the wild type single seeds were in the range of 200–400nmol glucose min−1 mg−1 protein, similar to levels reported in other cultivars of B. napus (Bones, 1990). Due to the complex situation of variability in MYR activity among single transgenic seeds, the MYR activity assay by the GOD-Perid method or the fast version of the GOD-Perid method was used as a tool for further characterizations. The MINELESS seeds which showed MYR activity in the range of 0–10nmol glucose min−1 mg−1 protein were processed further.
Immunoblot characterizations of protein extracts from MINELESS seeds using the 3D7 anti-MYR antibody showed the absence of all three bands of MYR isoenzymes found in B. napus (the 65, 70, and 75kDa classes of MYRs), while all these bands, as expected, were present in wild type protein extracts (Fig. 5A, B). MYRs are glycosylated dimeric proteins with subunit molecular weights in the range of 62–75kDa in plants (Bones and Rossiter, 1996). In wild type seed extracts, the presence of 65, 70, and 75kDa MYR isoenzymes is consistent with the occurrence of three different MYR isoenzymes in mature seeds of B. napus. These are encoded by the MB, MC, and MA gene family members, respectively (Lenman et al., 1990; Falk et al., 1995; Rask et al., 2000). Immunolocalization studies of wild type plants indicate the presence of isoenzymes in myrosin cells (Thangstad et al., 1990, 1991; Bones et al., 1991; Höglund et al., 1992; Kelly et al., 1998). The absence of the 65, 70, and 75kDa isoenzyme bands in MINELESS seed extracts provides direct evidence that the transgenic seeds are impaired in the synthesis and/or storage of MYR because of myrosin cell ablation. Therefore, it can be concluded that the Myr1.Bn1 promoter activates the expression of barnase in myrosin cells, thereby leading to their ablation.
Furthermore, immunoblot analysis of MBPs from wild type and MINELESS seeds indicates that the MBPs are present in both wild type and MINELESS seeds. It should be noted that MBPs are highly variable in expression (Taipalensuu et al., 1997). The presence of all MBPs in MINELESS seeds corresponds well with the results of a previous study by Eriksson et al. (2002), where immunohistochemical analysis showed that the MBPs are present in most cells, but not in myrosin cells. Further, Eriksson and co-workers have shown that the expression of MYR in seed myrosin cells of B. napus antisense plants was unaffected by the down-regulation of seed MBPs (Eriksson et al., 2002).
Two isoforms of ESP that have been characterized in B. napus have molecular masses of 39kDa and 35kDa (Bernardi et al., 2000; Foo et al., 2000), while in broccoli and cabbage ESP has been shown to be 37kDa and 43kDa (Matusheski et al., 2006). Consistent with these studies, two polypeptides with molecular masses of ~39kDa and 35kDa were detected in wild type B. napus seeds in the present study (Fig. 6D). Immunoblot analysis of MINELESS seeds using the same anti-ESP antibodies showed the absence of only the 35kDa polypeptide or of both the 39kDa and 35kDa polypeptides. This indicates a possible co-localization of ESP with MYR in seed myrosin cells. The co-localization of MYR and ESP in myrosin cells of Brassica seeds and cotyledons should be further confirmed with the use of other experimental approaches. Other possible explanations are that myrosin cells contain a factor that affects either ESP transcription and de novo ESP synthesis, or the generation of the 35kDa band from the 39kDa band (e.g. by proteolytic cleavage/processing). In A. thaliana, ESP has been reported to be localized to the epidermis of all above-ground organs except the anthers and in S-cells of the stem below the inflorescence, but not in myrosin cells (Burow et al., 2007b). It should, however, be noted that MYRs also have a different localization in A. thaliana compared with Brassica species (Andreasson et al., 2001; Husebye et al., 2002; Thangstad et al., 2004).
The presence of detectable levels of hydrolysed products in the GC-MS analysis (Fig. 6B) might indicate that the MINELESS seeds are not completely MYR free, but could have trace amounts (see also ‘Structural analysis’). However, these small amounts of hydrolysed products in the tested MINELESS seeds could also be due to thermal or other enzymatic degradation of GSL (Bones and Rossiter, 2006). Another possibility is that peroxisome-localized PEN2 proteins recently reported to have thioglucosidase activity are responsible for a low GSL-hydrolysing activity (Bednarek et al., 2009; Clay et al., 2009).
External addition of MYR to MINELESS seed extracts restored the capacity of MINELESS seed extract to degrade sinigrin, confirming that MYR is the major limiting factor for GSL hydrolysis in MINELESS. Since the degradation products, both in the wild type and in MINELESS, come from added sinigrin, one would expect almost the same levels of hydrolysis products from MINELESS (with added MYR) as were observed in wild type seed extracts. However, this assumption holds for the allyl nitrile and allyl isothiocyanate levels, but not for the 3,4-epithiobutane nitrile levels. The level of the latter is significantly higher in wild type seed extracts as compared with MINELESS seed extracts with or without addition of MYR (Fig. 6C). The activity assay used to hydrolyse sinigrin is designed to favour the formation of epithionitriles. A close to complete restoration of 3,4-epithiobutane nitrile production in MINELESS is therefore expected if ESP levels are similar to wild type levels. 3,4-Epithiobutane nitrile is not a major hydrolysis product from MINELESS extract degradation of sinigrin even after addition of MYR. It is suggested that this is due to the absence of one or both ESP isoforms in MINELESS tissue that was observed by immunoblot.
The GSL profile of MINELESS seeds is somewhat altered as compared with the parent cultivar Westar (wild type) (Fig. 7C), and affects both aliphatic and, to a lesser extent, indolic GSLs. Overall, the profiles resembled GSL profiles from previous studies performed with B. napus seeds (Fenwick and Heaney, 1983; Fenwick et al., 1983; Sang et al., 1984; Kraling et al., 1990; Daun and McGregor, 1991; Shahidi et al., 1997; Matthaus and Luftmann, 2000). Evidence of a rapid turnover of GSLs even in unchallenged plants has been reported (James and Rossiter, 1991; Rosa et al., 1994; Svanem et al., 1997; Thangstad et al., 2001; Petersen et al., 2002). The average GSL content of MINELESS seeds was increased (Fig. 7B), but varied considerably among single seeds (Fig. 8). The GSL level of the wild type was similar to that of its parent cultivar ‘Canola’ at ~10nmol GSL mg−1 seed (Downey, 1990). An interesting observation is that the GSL content showed variation in MINELESS seeds with up to 37nmol GSL mg−1 seed, which was above the level of Canola.
The elevated levels of GSLs in the MINELESS seeds might resemble an ongoing biosynthesis but an absence of in vivo catabolism/hydrolysis. In seeds of the Brassicaceae family, e.g. B. napus, S. alba, and Armoracia rusticana, a comparable correlation between MYR and GSL levels can be observed (Bones and Iversen, 1985; Li and Kushad, 2004). However a study on A. thaliana (Barth and Jander, 2006) has indicated that the developmental decreases in GSL content during senescence and germination occur independently of MYRs (TGG1 and TGG2). Various stresses, such as mechanical wounding (Bodnaryk, 1992) and insect and pathogen attack (Brader et al., 2001; Mewis et al., 2005; Mewis et al., 2006; Kim and Jander, 2007; Kusnierczyk et al., 2007, 2008; Ahuja et al., 2009; Bednarek et al., 2009; Clay et al., 2009) have been reported to induce alterations in the GSL profile and amount.
The targeted cell ablation strategy has been applied to genetically remove the MYR-containing myrosin cells from B. napus seeds. The production of MINELESS seeds is the first step on the way to reduce the anti-nutritional properties of rapeseed meal. In addition, the MINELESS plants, which lack the GSL–MYR defence, comprise an important model to study the plant defence system and other related functions. Further work will concentrate on the production and analysis of homozygous lines, and application of these seeds to study their nutritional quality. Studies of nutritional quality of the transgenic seeds, possible changes in secondary metabolic processes, and plant defence mechanisms are expected to yield new information, which will contribute to a better understanding of the GSL–MYR system.
Supplementary data are available at JXB online.
Figure S1. Specific myrosinase activity in seeds and cotyledons of the wild type and three transgenic lines.
Figure S2. Quantitative real-time PCR (qRT-PCR) analysis of glucosinolate metabolism genes (Myr1 and ESP) in 5-day-old cotyledons of the wild type and transgenic lines.
This work was supported by grants NFR 185173/V40, 175691/I10, and 184146/S10. We thank Dr Wilfred Keller (PBI, NRC, Canada) for providing Westar seeds, and Dr M Peferoen (Plant Genetic Systems Ghent, Belgium) for providing Barnase- and Barstar-encoding genes. We thank Drs Tore Brembu, Morten Lillemo, and Vidar Beisvåg for technical help with transformation of plants, Kjell Evjen for TEM sectioning, Dr Annavera De Felice for GSL analysis, Dr Jens Rohloff for analysis of GSL hydrolysis products, Dr Per Winge for designing qRT-PCR primers, and Bente Halvorsen for performing the qRT-PCR. Drs Lars Rask and Johan Meijer are acknowledged for providing 3D7 and 31:14 antibodies. The authors thank Nancy Bazilchuk and Drs Ralph Kissen and Anna Kusnierczyk for critical reading of the manuscript.