JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2443-2452; doi:10.1093/jxb/eri237
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RESEARCH PAPER |
Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich storage protein, in transgenic alfalfa but not in transgenic tobacco plants
1Plant Science Laboratory, Migal–Galilee Technology Center, PO Box 831, Kiryat Shmona, 11016, Israel
2Department of Agronomy and Natural Resources Department, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
3Tel-Hai Academic College, Upper Galilee 10120, Israel
* To whom correspondence should be addressed. Fax: +972 4 6944980. E-mail: rachel{at}migal.org.il
Received 7 December 2004; Accepted 28 May 2005
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Abstract |
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With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a ß-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine
-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others.
Key words:
Amino acids, cystathionine -synthase, forage plants, 15 kDa zein, methionine, methionine-rich storage protein, nutritional quality, transgenic plants
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Introduction |
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The nutritional value of plant proteins for humans and monogastric domestic animals is predominantly limited by their unbalanced essential amino acid composition. In this respect, methionine plays an important role being the major limiting essential amino acid in many crop plants, especially legumes (Muntz et al., 1997
; Tabe and Higgins, 1998). This limitation reduces wool growth on sheep, milk production by dairy animals, and meat quality (Pickering and Reis, 1993; Tabe et al., 1995; Xu et al., 1998). To meet the requirements of monogastric animal diets, methionine must be added either in a synthetic form, or supplied as methionine-rich forage plants. Moreover, ruminant animals must be supplied with methionine-rich proteins that are resistant to rumen proteolysis (Galili et al., 2002; Bagga et al., 2004).
Many efforts have attempted to improve the methionine content of vegetative tissues, with particular emphasis on forage legumes. The approach taken thus far has been based on the use of genes encoding methionine-rich seed storage proteins fused to a constitutive promoter or to leaf-specific promoters (Tabe and Higgins, 1998; Amir and Galili, 2003). Although most of these proteins were unstable in vegetative tissues (Wandelt et al., 1992; Ealing et al., 1994), some were stable when directed into the endoplasmic reticulum (ER) (Wandelt et al., 1992; Habben and Larkins, 1995; Khan et al., 1996; Christiansen et al., 2000; Tabe and Droux, 2001; Bagga et al., 2004). One of the stable proteins that naturally accumulates in ER-derived protein bodies is the 15 kDa zein (ß-zein) of corn. This protein has 11% methionine in its coding sequence and is stable in seed- and non-seed tissues of both monocotyledonous and dicotyledonous plants (Hoffman et al., 1987; Bagga et al., 1995, 1997, 2004; Bellucci et al., 2002). The 15 kDa zein is also resistant to digestion by rumen microorganisms (Bagga et al., 2004). Because of these attributes, the 15 kDa zein is an ideal candidate for raising the methionine content of forage plants to be used for ruminant animal feeding (Bagga et al., 2004).
However, expression of various methionine-rich proteins in vegetative tissues does not lead to sufficient elevation in methionine levels to meet the requirements for animal feeding. Apparently, the very low natural pool of free methionine in plants may limit the accumulation of methionine-rich proteins (Amir and Galili, 2003; Tabe and Higgins, 1998). Indeed, when such methionine-rich proteins were expressed in seeds, their accumulation was at the expense of other sulphur compounds or of other endogenous methionine-rich proteins (Jung et al., 1997; Muntz et al., 1997; Tabe and Droux, 2002; Hagan et al., 2003; Chiaiese et al., 2004).
The level of soluble methionine can be increased by overexpressing cystathionine -synthase (CGS), the first unique enzyme in the methionine biosynthesis pathway. Indeed, it has recently been reported that transgenic Arabidopsis, potato, tobacco, and alfalfa plants overexpressing the Arabidopsis CGS (AtCGS) have higher levels of soluble methionine (Hacham et al., 2002; Kim et al., 2002; Di et al., 2003; Avraham et al., 2005). Moreover, the level of methionine incorporated into proteins and of S-methylmethionine (SMM), the storage and mobile form of methionine, significantly increased in these transgenic tobacco and alfalfa plants (Hacham et al., 2002; Avraham et al., 2005). Transgenic tobacco, however, emitted from their leaves high amounts of dimethylsulphide, the catabolic product of methionine (Hacham et al., 2002), which was not detected in alfalfa plants. Notably, overexpression of plants CGS does not lead to higher level of methionine in all cases: Potato plants overexpressing their own CGS showed high transgene RNA level and 2.7-fold elevation in CGS activity, but the soluble methionine level remained unchanged (Kreft et al., 2003).
In this study, the role of soluble methionine in the accumulation of methionine-rich storage protein was examined by combining two approaches, namely, overexpression of methionine-rich storage protein and overexpression of CGS. To this end, the methionine-rich storage protein of 15 kDa zein was selected and combined with AtCGS. This strategy was applied to transgenic tobacco and alfalfa plants, as well as to a suspension of tobacco BY2 cells. It was found that soluble methionine limited the accumulation of the alien protein in transgenic alfalfa and BY2 cells but not in transgenic tobacco plants, demonstrating that, in addition to soluble methionine content, other factors are likely to be involved in the accumulation of 15 kDa zein in tobacco.
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Materials and methods |
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Constructing the plasmid for the expression of the maize ß-zein in plants
The maize ß-zein cDNA was PCR amplified from pMEZ (Bagga et al., 1995
), kindly donated by C Sengupta-Gopalan, New Mexico State University. Two primers were used, primer 1: 5'-CGTCTAGAGAACAGAACAGCATGAAGATG-3' and primer 2: 5'-TCCCCCGGGGTAG TAGGGCGGAATGGCAGC-3'. This zein fragment starts 11 bp upstream to the first ATG codon but lacks its natural stop codon. The PCR fragment was ligated to the PCR vector pGEMT (Promega, Madison, WI) to yield pGEMT-zein, and the nucleotide sequence of the amplified fragment was verified by DNA sequencing. The pGEMT-zein was then digested by XbaI and SmaI located in primers 1 and 2, respectively. The fragment was subcloned into a modified binary Ti plasmid, pZP111, digested by the same enzymes (Hadjukiewicz et al., 1994) between the 35S promoter of the CaMV and the Nos terminator, and in-frame to an epitope tag of 3x haemagglutinin (3HA), followed by a TGA stop codon (Tang et al., 2000) to yield pZP111-zein::3HA.
Plant transformation and selection
Tobacco plants cv. Samsung NN were transformed as previously described (Horsch et al., 1985). Transgenic plants were selected on Nitch (DuShefa) media containing 100 mg l–1 kanamycin. Alfalfa (Medicago sativa L.) cultivar SY plants were transformed as described (Galili et al., 2000) and regenerated on medium containing 100 mg l–1 kanamycin. Suspended cell cultures of tobacco (Nicotiana tabacum L. BY2 cells) were transformed as described (Takeda et al., 1992). The transformed BY2 cells were grown on Marashige and Skoog (MS) medium containing 100 µg ml–1 kanamycin and 250 µg ml–1 cephatoxime (Takeda et al., 1992).
Protein isolation and immunoblot analysis
Leaf samples (100 mg) of wild-type and transgenic plants expressing the ß-zein::3HA protein were homogenized by mortar and pestle in a buffer containing 25 mM TRIS–HCl, pH 7.5, 2 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulphonyl fluoride (PMSF) at 4 °C. After 25 min of centrifugation at 16 000 g at 4 °C, total soluble protein was determined in the supernatant using the Bradford assay (Bio-Rad). The zein-3HA protein was extracted from the pellet by incubation in 70% ethanol containing 1% ß-mercaptoethanol at 60 °C for 30 min. Alcohol-soluble protein samples were dried in a Speed Vac and solubilized in SDS sample buffer containing 5% ß-mercaptoethanol. For immunoblot analysis, ethanol-soluble protein samples equivalent to a known amount of the PBS-soluble protein extract (4 µg) were fractionated on 12% (w/v) SDS–PAGE (Laemmli, 1970). Fractionated proteins were transferred to polyvinyldifluoride (PVDF) membrane, stained with Ponceau-S, and reacted with commercial anti-3HA monoclonal antibodies (Roche, Basel) using the ECL kit (Amersham), as recommended by the manufacturer.
Isolation of RNA and northern blot analysis
Total RNA was extracted from 100 mg leaves using the Tri-Reagent (Sigma) according to the protocol provided by the manufacturer. RNA samples (20 µg) were subjected to electrophoresis in 1% agarose gel containing 2.2 M formaldehyde and 50 mM MOPS, pH 7.0, and transferred onto a Hybond N membrane (Amersham). Blots were hybridized for 12 h at 65 °C with probes labelled with -32P dCTP by the Rediprime kit (Amersham) using the PCR product of the ß-zein as a probe. For detection of internal loading, membranes were rehybridized with the 18S ribosomal RNA probe.
GC-MS analysis of soluble amino acid content
For analysis of soluble amino acid content, replicate samples of 100 mg were ground to powder and extracted in 2400 µl methanol. Norleucine (4.6 µl of 2 mg ml–1) was added as an internal standard. After a vortex, the mixture was extracted for 15 min at 70 °C and mixed with 750 µl water. To separate the polar phase, 375 µl chloroform was added, the mixture was briefly mixed by a vortex and separated by 15 min centrifugation at 2200 g. Three hundred µl of the upper polar phase of each sample were reduced to dryness under vacuum, and then dissolved and treated for 2 h with 40 µl 20 mg ml–1 methoxyamine hydrochloride in pyridine, followed by derivatization for 30 min in N-methyl-N(trimethylsilyl)-trifluoroacetamide at 37 °C with vigorous shaking. Sample volumes of 1 µl were injected into a GC-MS system with a split ratio of 1:1. Along with the samples, amino acid standards of 200, 100, 50, and 25 µM were injected to establish quantification curves. The GC-MS system included an HP6890 autosampler injector, HP5890 series II plus gas chromatograph, and an HP5972 series selective detector mass spectrometer (Hewlett Packard, USA). GC was performed on a 30 m Rtx-5SIL MS column with 0.25 mm inner diameter and 0.25 µm film thickness (Restek, USA, Cat no. 12723-124). Injection temperature was 230 °C, the interface set to 250 °C. Helium carrier gas flow was 1 ml min–1, and the temperature was set to 5 min heating at 70 °C, followed by a 5 °C min–1 increase in temperature to 310 °C, with an additional 1 min heating at 310 °C. Prior to injection of the next sample, the system was equilibrated for 6 min at 70 °C. Mass spectra was recorded at two scans s–1, with a m/z 50–600 scanning range. The mean quantity of each amino acid was calculated from calibration curves of standards, taking norlucine as an additional reference.
Analysis of methionine metabolites
The level of dimethyldisulphide was examined using GC-MS (Hacham et al., 2002). For the determination of methionine levels in water- and ethanol-soluble proteins, proteins were extracted as described above and their level was determined using the Bradford method. Forty µg proteins were then hydrolysed in 0.3 ml 6 N HCl at 110 °C for 22 h under vacuum. Samples (4 µg) of the hydrolysed proteins were analysed by HPLC (Dionex, Bio LC Amino Acid Analyser).
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Results |
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Production of transgenic tobacco and alfalfa plants expressing the ß-zein::3HA gene
The ß-zein::3HA gene was overexpressed under the control of the constitutive promoter of the cauliflower mosaic virus 35S in transgenic tobacco and alfalfa plants. Thirty independent kanamycin-resistant tobacco lines and 18 alfalfa lines were obtained. The expression level of the 15 kDa zein::3HA was determined by western blot analysis using anti-3HA antibodies (data not shown). As expected, an 18 kDa band representing the 15 kDa zein and the 3HA peptide was apparent. Two tobacco lines, Z4 and Z5, and two alfalfa lines, L53 and L54, showing the highest expression level of the 15 kDa zein::3HA were selected for further studies. No morphological differences were noticed between the control, non-transformed tobacco, and alfalfa plants, and the corresponding transgenic plants expressing the 15 kDa zein::3HA.
Following selfing of the two selected T0 tobacco lines, the obtained T1 progeny segregated on kanamycin-containing MS medium at a 3:1 ratio, indicating that the transgene behaves as a single dominant locus. Alfalfa, a tetraploid plant, rarely produces a second generation due to self-incompatibility, and hence, T0 alfalfa plants were studied.
Soluble methionine limits the 15 kDa zein::3HA accumulation in transgenic alfalfa and BY2 cells but not in transgenic tobacco plants
To study the effect of soluble methionine on the 15 kDa zein::3HA accumulation, the transgenic tobacco and alfalfa plants were first irrigated with methionine. Homozygous transgenic tobacco plants were irrigated for 5 d with 5, 10, and 20 mM methionine, while control plants were irrigated with distilled water. As shown in Fig. 1A, the exogenously applied soluble methionine hardly affected the accumulation of the 15 kDa zein::3HA in the two transgenic tobacco lines (shown for fully expanded leaves irrigated with 10 mM methionine). However, following irrigation of the two transgenic alfalfa lines, L53 and L54, with 2 mM methionine for 2 d, the expression level of the 15 kDa zein::3HA was significantly increased (5- and 8-fold, respectively) (Fig. 1B).
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To shed light on the differential response of transgenic tobacco and alfalfa plants to methionine, tobacco BY2 suspension cells were used in which methionine can penetrate the cells more efficiently than in the whole plant system. BY2 cells were transformed with the ß-zein::3HA construct. As shown in Fig. 1C, the addition of 1 mM methionine to 5-d-old transgenic BY2 cultures 24 h before harvesting significantly increased (
8-fold) the expression level of the 15 kDa ß-zein::3HA. This finding not only demonstrated that methionine penetrates into these cells, but it also suggests that, in some tissues like the meristem or wound tissue, methionine may limit the 15 kDa zein::3HA accumulation in transgenic tobacco plants as well.
Co-expression of the 15 kDa zein::3HA and AtCGS in transgenic tobacco plants does not significantly affect the accumulation of the 15 kDa zein::3HA
Considering the response of BY2 culture cells, it was examined next whether the inability of methionine to enhance the accumulation of the 15 kDa zein::3HA in leaves of transgenic tobacco plants stemmed from its failure to penetrate the root system or to be transferred efficiently to leaves. To ascertain elevated endogenous levels of methionine, T1 homozygous transgenic tobacco plants expressing the 15 kDa ß-zein::3HA (lines Z4 and Z5) were crossed with two homozygous plants overexpressing AtCGS that exhibit high levels of endogenous methionine and SMM in their leaves (Hacham et al., 2002). For these crosses, transgenic line F4 was used which lacks a morphologic phenotype, and transgenic line F30 which exhibits a phenotype similar to plants expressing the truncated form of AtCGS (Hacham et al., 2002). The latter line had significantly higher levels of methionine and dimethylsulphide, the catabolic product of methionine, 33-fold and 20-fold than wild-type plants, respectively (Table 1).
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The expression level of the 15 kDa zein::3HA was elevated only slightly and non-significantly in F1 plants Z5/F4 and Z4/F4 and in Z5/F30 and Z4/F30 compared with heterozygous plants expressing only the 15 kDa ß-zein::3HA (shown for Z5/F4 and Z5/F30, Fig. 2A).
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Methionine content is reduced in tobacco plants co-expressing the 15 kDa zein::3HA and AtCGS compared with plants expressing AtCGS alone
Next, the levels of soluble methionine, dimethylsulphide as well as of methionine in the ethanol- and water-soluble protein fractions were determined in the transgenic tobacco plants. The soluble methionine level was significantly reduced in plants co-expressing both alien genes compared with those expressing AtCGS alone (Fig. 3A; Table 1). This suggests that the level of the 15 kDa zein::3HA was apparently increased in these plants, an elevation that apparently was not sufficiently detected using western blot analysis (Fig. 2A). To clarify this issue further, the ethanol-soluble fraction was separated on SDS–PAGE and stained by Coomassie blue (Fig. 4A, upper panel); for controls, water-soluble proteins of the same extracts were also loaded and stained (Fig. 4A, lower panel). The total ethanol fraction was about ten times lower in tobacco than in alfalfa plants, but in both types of transgenic plants the major band (the 15 kDa zein::3HA) in the ethanol-soluble fraction showed higher intensity in plants co-expressing the two alien genes compared with plants expressing only the 15 kDa zein::3HA (Fig. 4A, upper panel). To study whether this elevation affected the methionine level in the ethanol-soluble fraction, this fraction was subjected to protein hydrolysis. As shown in Table 1, the methionine level in the two protein fractions was not significantly different when the two sets of transgenic plants were compared. Moreover, the methionine level in the ethanol-soluble protein fraction was not affected in transgenic plants expressing the 15 kDa zein::3HA relative to wild-type plants. This suggests that the protein hydrolysis method yielded unreliable results, when a small protein fraction, like the ethanol fraction of plant cells, was used for the analysis.
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Notably, transgenic plants derived from crosses between F30 and Z4 or Z5 remained phenotypically similar to plant F30, and they still emitted a high level of dimethylsulphide compared with wild-type plants (Table 1). These results indicate that the excess endogenous methionine in these transgenic tobacco plants was still accompanied by the abnormal phenotype and was degraded into dimethylsulphide, without being incorporated to high levels into the 15 kDa zein::3HA protein.
Elevated endogenous methionine significantly enhances the 15 kDa zein::3HA level in transgenic alfalfa plants
Transgenic alfalfa plants expressing the ß-zein::3HA gene (lines L53 and L54) were crossed with transgenic plants expressing AtCGS (line L19), in which soluble methionine and SMM are found at a high level (Avraham et al., 2005). A large number of independent progeny was thus obtained. However, due to the self-incompatibility that occurs naturally in alfalfa, only two F1 plants were found to express both foreign genes (L54/L19/1 and L54/L19/2, Fig. 2B). In these plants, the level of the 15 kDa zein::3HA was significantly elevated (7- and 10-fold) compared with transgenic alfalfa plants expressing only the 15 kDa zein::3HA (Fig. 2B). Significantly stronger bands were also found when the ethanol-soluble fraction derived from plants expressing both alien genes was subjected to SDS–PAGE fractionation (Fig. 4B, upper panel). To determine whether the elevation in 15 kDa zein::3HA affected the level of soluble methionine or of methionine found in the water-soluble protein fraction, the transgenic alfalfa plants were analysed as described above. Soluble methionine decreased significantly in plants expressing both alien genes compared with those expressing AtCGS alone (Fig. 3B; Table 2). However, this reduction did not affect the level of methionine in the water-soluble protein fraction (Table 2; Fig. 4B). Therefore, the results suggest that the higher level of soluble methionine enhances the accumulation of the 15 kDa zein::3HA in alfalfa.
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The level of the 15 kDa zein::3HA in transgenic tobacco plants is controlled post- transcriptionally
The results described thus far show that soluble methionine limits the level of 15 kDa zein::3HA accumulation in transgenic alfalfa but far less in transgenic tobacco. Two factors can regulate the 15 kDa zein::3HA accumulation in transgenic tobacco. First, the methionine level may be high enough to support the 15 kDa zein::3HA accumulation, so that excess methionine will not increase its level any further. Second, some as yet unknown factor/s may regulate its transcript or its protein accumulation. To explore these possibilities, the 15 kDa zein::3HA gene dosage was doubled in transgenic tobacco by producing plants homozygous for this gene and then the protein and the transcript levels in homozygous versus heterozygous plants were compared. Although the transcript level almost doubled in homozygous relative to heterozygous transgenic tobacco plants (Fig. 5A), only minor changes were noted at the protein level (Fig. 5B). This suggests that post-transcriptional or post-translational regulation, rather than the soluble methionine content, are likely to control the accumulation of this foreign protein in transgenic tobacco plants.
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Discussion |
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As part of a long-term goal to increase plants methionine content and to examine factors involved in methionine accumulation, the effect of soluble methionine on the accumulation of the 15 kDa-zein::3HA, a methionine-rich protein, in transgenic tobacco and alfalfa plants was investigated here.
Overexpressing methionine-rich storage proteins in leaves has been one of the main approaches in the attempt to increase methionine content in forage plants (Amir and Galili, 2003, and references therein). Although this approach led to an increase in total methionine in seeds (Altenbach et al., 1989, 1992; Molvig et al., 1997; Muntz et al., 1997; Tabe and Higgins, 1998; Lee et al., 2003), such elevation fell short of supporting the demands for human and animal feeding. Detailed examination revealed that the expression of methionine-rich storage proteins was at the expense of other sulphur compounds, particularly endogenous sulphur-rich compounds and methionine-rich proteins (Muntz et al., 1997; Tabe and Droux, 2002; Hagan et al., 2003; Chiaiese et al., 2004). So, for example, cotyledons in transgenic lupin expressing the sunflower 2S albumin contained less free methionine, cysteine, and glutathione than control plants and also exhibited a drop in the level of endogenous sulphur-rich proteins (Tabe and Droux, 2002). A similar apparent reallocation of sulphur from endogenous proteins to the heterologous sulphur-rich protein has been reported in transgenic corn expressing a sulphur-rich zein (Anthony et al., 1997) and in transgenic soybean expressing the Brazil nut 2S albumin (Jung et al., 1997). These findings indicate that the available soluble cysteine and methionine may limit the accumulation of foreign sulphur-rich proteins in transgenic plants.
In the attempt to enhance the accumulation of both foreign proteins as well as the endogenous methionine-rich proteins without reducing the level of other sulphur compounds, the suggestion in this study is to co-express in transgenic plants methionine-rich storage proteins side by side with a key enzyme in the methionine biosynthesis pathway. Using this approach, the authors had previously succeeded in elevating the lysine content in leaves of transgenic tobacco plants by expressing both the lysine-rich protein of soybean vegetative storage protein ß subunit (S-VSPß) and the bacterial feedback-insensitive dihydrodipicolinate synthase (Guenoune et al., 2003). In plants expressing both foreign genes, the level of S-VSPß was significantly increased compared with plants expressing S-VSPß alone. In addition, total lysine level (soluble and protein-bound) in these transgenic tobacco plants increased by 30% above that of wild-type plants (Guenoune et al., 2003).
Unlike lysine, which mainly serves protein synthesis, methionine is also required as a precursor for S-adenosyl-methionine (SAM), a methyl donor and a substrate for the synthesis of a number of essential metabolites including polyamines, biotin, and ethylene (reviewed in Droux et al., 2000; Amir and Galili, 2003; Hesse and Hofgen, 2004). Thus, in leaves, the competition between protein synthesis and SAM synthesis can play a major role in regulating the soluble methionine level required for protein synthesis. However, an increase in soluble methionine, brought about by the overexpression of AtCGS in tobacco plants, contributed to both methionine within endogenous proteins and to SAM metabolites (Hacham et al., 2002). Moreover, the level of soluble methionine affects the nature of the synthesized proteins by modifying the ratio between methionine-rich and -poor proteins. This regulation occurs through transcriptional and post-transcriptional processes (Beach et al., 1985; Naito et al., 1994; Hirai et al., 1995; Hagan et al., 2003).
Major differences in the composition of water-soluble seed storage proteins have been reported recently in transgenic rice seeds expressing a methionine-rich storage protein (Hagan et al., 2003). The researches suggested that this response is mediated by a signal transduction pathway that normally modulates seed storage protein composition following environmental fluctuations in sulphur availability (Hagan et al., 2003). Such differences in protein composition were not detected in transgenic tobacco and alfalfa proteins that expressed the 15 kDa zein (Fig. 4). The distinct response of rice relative to tobacco/alfalfa plants may be due to inherent differences between seeds that have major storage proteins and vegetative tissues where the main protein is Rubisco and numerous other proteins having lower expression level. The protein pattern in this fraction, which accounts for 80% of the total proteins (Galili et al., 2000), is probably altered in transgenic tobacco/alfalfa plants overexpressing AtCGS, as these plants demonstrated higher levels of methionine incorporated into proteins (Tables 1, 2). However, these changes in protein composition were not detected in this system (Fig. 4).
Elevated levels of specific soluble amino acids contribute to their higher content in those endogenous proteins that are enriched with them (Frankard et al., 1991; Habben and Larkins, 1995; Heremans and Jacobs, 1997; Galili et al., 2000), as found for methionine (this study, and Hacham et al., 2002; Avraham et al., 2005). The elevation in endogenous methionine-rich proteins can supply adequate amounts of methionine to feed monogastric animals without resorting to additional synthetic methionine (Avraham et al., 2005). However, for ruminant animals, methionine must be supplied in the form of proteins that are resistant to rumen proteolysis such as the 15 kDa zein studied here (Galili et al., 2002; Bagga et al., 2004). Therefore, it is proposed to examine the effect of soluble methionine on a foreign methionine-rich storage protein, which is insensitive to ruminant degradation.
To study the effects of soluble methionine on the15 kDa zein::3HA accumulation, methionine was first added to the growth medium of transgenic plants. In a different approach, these plants were crossed with plants expressing the AtCGS, having higher endogenous levels of methionine. The expression of 15 kDa zein::3HA was then monitored. In tobacco plants, results showed that when methionine was added to the medium the expression level of 15 kDa zein::3HA was not affected (Fig. 1A). However, in plants co-expressing 15 kDa zein::3HA/AtCGS, the expression level of 15 kDa zein::3HA increased slightly, although non-significantly (Fig. 2A). This slight elevation was accompanied by a reduction in soluble methionine and increased band intensity in the ethanol-soluble protein fraction compared with plants expressing AtCGS alone (Figs 3A, 4A, respectively). This supports the observation that the level of 15 kDa zein::3HA indeed increased, and that soluble methionine has been incorporated into this protein. Nonetheless, this effect is less conspicuous compared with that found in alfalfa. In alfalfa, both the exogenously applied methionine and the higher endogenous methionine content significantly increased the expression level of the 15 kDa zein::3HA in the transgenic plants (Figs 1B, 2B). In addition, plants co-expressing the 15 kDa zein::3HA/AtCGS possessed significantly lower soluble methionine than plants expressing only the AtCGS (Fig. 3B), while the expression level of 15 kDa zein::3HA has been significantly enhanced (Fig. 2B) and the total ethanol-soluble proteins was elevated (Fig. 4B). Together, these results suggest that the higher level of soluble methionine enhances the accumulation of the 15 kDa zein::3HA, a phenomenon clearly demonstrated for alfalfa and to a much lesser extent for tobacco.
Why tobacco and alfalfa respond differently to the higher methionine content is not yet clear. Possibly, the extremely low level of methionine normally found in legumes (Tabe and Higgins, 1998) may limit the 15 kDa zein::3HA accumulation in alfalfa. On the other hand, in tobacco, where the level of methionine may be high enough to support such accumulation, any further increase in methionine would hardly enhance the 15 kDa zein::3HA level. Alternatively, the differences between tobacco and alfalfa may lie in factors that regulate the steady-state transcript or protein levels of zein in tobacco, which apparently may not exist in alfalfa. To distinguish between these possibilities, the dosage of the ß-zein gene was doubled in tobacco. If the soluble methionine level had been high enough in these plants, it is expected that the expression of the 15 kDa zein::3HA protein would double in homozygous compared with heterozygous plants; however, if post- transcriptional factors regulate the steady-state level of the 15 kDa zein::3HA, one expects to detect no difference between the two sets of plants at the transcript or at the protein levels. It was found that the 15 kDa zein::3HA transcript level doubled in homozygous plants, but the protein level was only slightly affected (Fig. 5). Because, in tobacco, a higher level of methionine does not significantly affect the 15 kDa zein level, it is suggested that the soluble methionine level is not the factor controlling the 15 kDa zein::3HA accumulation in transgenic tobacco plants. It is proposed that the 15 kDa zein::3HA level is regulated post-transcriptionally or post-translationally by some, as yet, unknown factors. Although methionine may not affect the level of the 15 kDa zein in tobacco, the possibility cannot be ruled out that differences between alfalfa and tobacco may also reflect differences in tRNAmet that can limit the synthesis of the 15 kDa-zein::3HA in alfalfa but not in tobacco.
Factors that regulate the accumulation of the 15 kDa zein may be found in the ER-derived protein bodies, such as the BiP chaperone, whose expression is enhanced in plants expressing the15 kDa-zein::3HA (Bagga et al., 1997; Bellucci et al., 2000, 2002). Although ER-protein bodies are considered a favourable environment for the accumulation of sulphur-rich proteins, these proteins do not account for more than 0.8% of the total proteins (Tabe et al., 1995; Khan et al., 1996; Sharma et al., 1998), indicating that their level was tightly controlled in the ER. For comparison, the highest level of expression in plants was found in the gene encoding phytase, reaching 14.4% of the total leaf soluble proteins (Verwoerd et al., 1995).
Post-translational regulation probably does not affect the15 kDa-zein::3HA stability in tobacco plants as this protein was found to be stable in vegetative tissues and in seeds of transgenic plants and remained intact even during the germination of transgenic tobacco seeds (Hoffman et al., 1987; Bagga et al., 1995). It was found, too, that this protein, unlike to native proteins, remained stable and was not degraded in old leaves of transgenic plants expressing this gene (data not shown). Nevertheless, further studies are needed to identify the factor/s that regulate the 15 kDa zein::3HA accumulation in tobacco, although most probably not in alfalfa where the zein protein is able to accumulate.
The present study shows that methionine may limit the accumulation of the 15 kDa zein::3HA in some plant species, but far less in others. To reveal the potential of improving the nutritional quality of transgenic plants by elevating the methionine content, one should express the methionine-rich storage protein and concomitantly irrigate the plants with methionine. Such an approach may indicate the potential benefits that can be derived from expressing enzymes like CGS with the consequent increase in soluble methionine. Co-expression of methionine ‘sink’ proteins combined with high levels of soluble methionine can enhance significantly the level of the foreign methionine-rich protein, as found here for transgenic alfalfa plants.
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Acknowledgements |
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We deeply thank Professor C Sengupta-Gopalan, New Mexico State University, for providing the maize ß-zein cDNA. This research was supported by the European FP5 (QLRT-2000-00103) project and by the Chief Scientist of the Ministry of Agriculture, Israel grant no. 868-0205-01.
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Footnotes |
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Abbreviations: CGS, cystathionine
-synthase; AtCGS, Arabidopsis cystathionine -synthase; 3HA, 3x haemagglutinin; SMM, S-methylmethionine; ER, endoplasmic reticulum. |
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