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Journal of Experimental Botany, Vol. 53, No. 376, pp. 1867-1870, September 1, 2002
© 2002 Oxford University Press

Combined expression of S-VSP{alpha} in two different organelles enhances its accumulation and total lysine production in leaves of transgenic tobacco plants

Received 11 January 2002; Accepted 3 June 2002

Dana Guenoune1, Rachel Amir2, Hanna Badani1, Shmuel Wolf3 and Shmuel Galili4,1

1 Agronomy and Natural Resources Department, Agricultural Research Organization, The Volcani Center, POB 6, Bet Dagan 50250, Israel
2 Department of Plant Physiology, Migal Technological Center, Kiryat Shmona 12100, Israel
3 The Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel

4 To whom correspondence should be addressed. Fax: +972 3 9669642. E-mail: vclidg{at}netvision.net.il


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Soybean vegetative storage proteins (S-VSPs) accumulate to high levels in vacuoles of both wild types and heterologous plants. Here it is shown that directing S-VSP{alpha} to two different organelles—chloroplasts and vacuoles—in a single transgenic plant significantly increased its accumulation. Accumulation of S-VSP{alpha} in heterologous plants correlated with total soluble lysine. Using this approach with essential amino-acid-rich transgene proteins may lead to a breakthrough in improving plant nutritional quality.

Key words: Key words: Chloroplasts, gene expression, improved nutritional quality, soybean vegetative storage proteins, total lysine content, transgenic tobacco plants.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
Improving the nutritional quality of plants was recently approached by expressing genes encoding lysine- or methionine-rich seed storage proteins that accumulate to high levels (Galili et al., 2002). These essential amino-acid-rich seed storage proteins accumulate to high levels when expressed in seed tissues, resulting in a significant improvement of seed nutritional quality (Molvig et al., 1997; Muntz et al., 1998). Expressing these proteins in vegetative tissues by fusing them to the strong constitutive cauliflower mosaic virus (CaMV) 35S promoter resulted in only a minor success (Galili et al., 2002). These proteins, which naturally accumulate in vacuolar-derived protein bodies, failed to accumulate inside the highly lytic vacuoles of vegetative tissues. This problem was partially overcome by directing the accumulation of these proteins to the endoplasmic reticulum (ER) (Galili et al., 2002), or by utilizing genes encoding storage proteins that naturally accumulate in ER-derived protein bodies (Bagga et al., 1995). The potential of utilizing genes coding for vegetative storage proteins of soybean (S-VSPs) to improve the nutritional quality of vegetative tissues has recently been demonstrated (Guenoune et al., 1999). S-VSPs contain about 7% lysine and accumulate to high levels inside vacuoles of vegetative tissues of both wild-type (WT) soybean (Staswick, 1994) and transgenic tobacco plants (Guenoune et al., 1999). Furthermore, as the accumulation of S-VSP{alpha} was not affected by leaf age, the encoding genes may be suitable candidates for improving the nutritional quality of forage crops. In view of problems encountered in the accumulation of various heterologous proteins in vegetative tissues, the aim of this study was to examine the capability of the S-VSPs to accumulate inside the chloroplast as a storage organelle. In addition, the possibility was examined of further increasing S-VSPs accumulation by directing them to more than one organelle in the same plant.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Plant material
The plant material used in this study consisted of Nicotiana tabacum L. cv. Samsun NN, soybean (Glycine max Merr.), and the following transgenic tobacco plants: line CE20, expressing S-VSP{alpha} inside the chloroplasts and line W2, expressing S-VSP{alpha} inside the vacuoles (both exhibiting the highest levels of transgene accumulation). Crosses were made between transgenic tobacco plants lines CE20 and W2 to form line CE20/W2. Plants were grown in a controlled environment chamber with a 14 h day length and 28/24 °C day/night temperature. Pots were watered with a complete nutrient solution according to Johnson et al. (1957). All plant material was harvested mid-morning and immediately frozen at –70 °C until use.

Construction of chimeric genes and plant transformation
In order to direct the S-VSP{alpha} to chloroplasts, the S-VSP{alpha} encoding DNA sequence of the plasmid pKSH1 (Mason et al., 1988) was amplified by polymerase chain reaction (PCR) using PWO DNA polymerase (Boehringer) to form an in-frame SphI site. This site substituted the first alanine codon GCC of the mature S-VSP{alpha} protein (Mason et al., 1988) by a methionine translation initiation codon ATG. The primers used were 5'-AGCATGCGTACTCCGGAGGTGAAATGC-3' and 5'-AGGAACTACTGAATGTAGTACAG-3'. The amplified fragment was cloned into the SmaI site of pBluescript KS+ (Stratagene), and this PCR fragment was subcloned into the SphI–SacI sites of pCE vector to form pCE20. pCE vector is a pBluescript SK+ derivative (Stratagene) plasmid containing a PUC 18 KpnI–HindIII fragment (that includes the CaMV 35S promoter, the {Omega} translation enhancer (Gallie et al., 1989), the pea rbcS-3A transit peptide (Fluhr et al., 1986) between the BamHI–SphI sites), and an octopin synthase terminator (Grave et al., 1983) between the PstI–SpeI sites. Finally, a SmaI–SacI fragment of pCE20 was subcloned into the SmaI–SacI site of pGTV–KAN binary Ti plasmid (Detlef et al., 1992) to form p104CE20. Tobacco plants Nicotiana tabacum L. cv. Samsun NN were transformed by the leaf disc protocol (Horsch et al., 1985) with Agrobacterium tumefaciens carrying the binary pBIN234 plasmid containing the S-VSP{alpha} coding sequence fused to the CaMV 35S promoter to form transgenic line W2 (Guenoune et al., 1999), and p104CE20 (see above) to form line CE20. Thirty independent T0 transgenic plants expressing each of these chimeric constructs were selected on 100 mg l–1 kanamycin sulphate and transferred to ‘Jiffy 7’ peat pellets (Soli, Israel) for establishment. Plants were transferred to 3.0 l pots after about 1 month.

Protein analysis
All methodologies used in this study, including protein extraction, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, Coomassie Brilliant Blue staining, and quantitative estimation of the S-VSP{alpha} band were as previously described (Guenoune et al., 1999). All experimental values were based on at least three independent plants of each line and at least three replicates were run for each plant. All statistic adaptations were done by F-test analysis using the JMP version 3.1.5 program.

Determination of total soluble lysine (including soluble lysine and lysine content in soluble proteins)
Total free amino acids and soluble proteins were extracted from 100 mg tobacco leaf samples and 40 µg were used to analyse the amino acid composition as previously described (Karchi et al., 1993).


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Stability of a transgene protein is an important limiting factor in many applications in which genetic engineering is used for plant improvement. Several studies showed that the accumulation level of transgene proteins in vegetative tissues is greatly influenced by their subcellular location (Wandelt et al., 1992). Until now, almost all studies examined the ER, the apoplast or the cytosol as the target organelle for transgene protein accumulation. The ability to accumulate heterologous proteins in the chloroplasts was examined only recently (Dai et al., 2000), but, it appears, however, that the accumulation depends mainly on the protein structure and composition. Thus, the potential of using chloroplasts as a storage organelle for S-VSP{alpha} was tested here by transforming tobacco plants with p104CE20 plasmid to form CE20 T0 plants.

Western blot analyses of these plants revealed a protein band of about 24 kDa (Fig. 1A) that was not detected in untransformed tobacco plants (Fig. 1A, lane N). As expected, the transgene protein of line CE20 migrated in SDS-PAGE faster than the wild-type S-VSP{alpha} of soybean (Fig. 1A, lane S), because of the cleavage of the transit peptide during its import into the chloroplasts (Dobberstein et al., 1977; Schmidt et al., 1981) and the lack of glycosylation that occurred only in the endomembrane system.



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  		Fig. 1. Effect of leaf age on accumulation of S-VSP{alpha} in transgenic tobacco plants. Leaf protein extracts (20 µg) were separated on SDS–PAGE and analysed by Western blot using anti S-VSPs antibodies. (A) T0 plant of line CE20 expressing S-VSP{alpha} inside its chloroplasts. (B) F1 plant derived from the cross between transgenic tobacco plants of line CE20 and line W2 (expressing S-VSP{alpha} inside its vacuoles). N, untransformed tobacco plants; S, WT soybean; Lanes 1, 4, 10, and 18 represent leaf number from the top of the plants. The numbers on the left represent molecular weight size in kDa.

 
In order to improve the nutritional quality of forage plants, the transgene protein used must accumulate to high levels in leaves of the whole plant. To study the effect of leaf age on the accumulation of S-VSP{alpha} in the chloroplasts, total soluble proteins were extracted from the youngest (No. 1) to the oldest (No. 18) leaf of 5-month-old primary transformant transgenic tobacco line CE20. The level of S-VSP{alpha} relative to the total soluble leaf proteins was stable from leaf Nos 1 to 10 (3–3.7%) and slightly reduced in the older leaves, declining to a value of about 2.3% in leaf No. 18 (Fig. 2, line CE20). The level of S-VSP{alpha} relative to the total soluble leaf proteins in line W2, in which S-VSP{alpha} accumulates in the vacuole, remained stable with leaf age (Fig. 2, line W2) as previously described (Guenoune et al., 1999). In most cases, the level of S-VSP{alpha} in line CE20 was slightly, but not significantly, higher than in line W2 (Fig. 2, lines CE20 and W2). Similarly, Dai et al. (2000) showed that the endoglucanase protein (E1) was expressed inside the chloroplasts where its expression was significantly higher compared to its expression inside the vacuoles, suggesting that the chloroplast may be an additional candidate storage organelle for nuclear encoded transgene proteins.



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  		Fig. 2. Relative levels of S-VSP{alpha} in leaves of different age in several transgenic tobacco lines. Protein extracts (20 µg) were separated on SDS-PAGE and stained with Coomassie Brilliant Blue, followed by scanning the stained gels. W2, line expressing S-VSP{alpha} inside the vacuoles; CE20, line expressing S-VSP{alpha} inside the chloroplasts; CE20/W2, F1 plants derived from the cross between lines CE20 and W2. S-VSP{alpha} level in line CE20/W2 is represented by stacked bars in which the lower and upper bars represent the relative levels of S-VSP{alpha} accumulated inside the vacuoles and the chloroplasts, respectively. The level of S-VSP{alpha} in the indicated leaves of the transgenic plants (mean of triplicate determinations) is plotted relative to the total soluble protein of each leaf tested. Values represent the means of three measurements (±SE).

 
Although chloroplasts are suitable storage organelles for S-VSP{alpha} accumulation, the total S-VSP{alpha} was not higher than the level obtained when this protein was targeted to the vacuoles. It was tested, therefore, whether directing a transgene protein, such as S-VSP{alpha}, to more than one organelle in the same plant may be a breakthrough to increase its accumulation further. For this purpose, transformants of lines CE20 and W2 were crossed to form line CE20/W2. Western blot analysis of these F1 plants revealed a pattern of two bands, indicating that S-VSP{alpha} transgene proteins accumulated in both organelles (Fig. 1B). The upper, 27 kDa band, which co-migrates with the S-VSP{alpha} of soybean, corresponds to the vacuolar S-VSP{alpha} protein and the lower, 24 kDa band, corresponds to the chloroplast S-VSP{alpha} protein. Both S-VSP{alpha} protein bands migrated in SDS-PAGE identically to their analogues in lines CE20 and W2 (the latter are not shown). The effect of leaf age on the accumulation of S-VSP{alpha} in these F1 plants is shown in Fig. 2 (line CE20/W2). The difference in the molecular weight between the two S-VSP{alpha} transgene proteins in line CE20/W2 enabled the accumulation of each protein to be followed separately. In all leaves tested, except for leaf No. 18, the accumulated level of the S-VSP{alpha} in the F1 line CE20/W2 was similar to the sum of the S-VSP{alpha} levels obtained in lines CE20 and W2 (Fig. 2). In leaf No. 18 of line CE20/W2, however, significantly higher S-VSP{alpha} levels were obtained compared to the sum of the S-VSP{alpha} levels obtained in lines CE20 and W2. The results indicate that the accumulation of S-VSP{alpha} in one organelle is not affected by its accumulation in an additional organelle. The accumulation of S-VSP{alpha} was also accompanied by an elevation in total soluble lysine (including soluble lysine and lysine content in the soluble protein fraction). Total soluble lysine was significantly higher in these transgenic lines compared to untransformed tobacco plants, increasing by about 7% in line W2, 10% in line CE20 and 17% in line CE20/W2 (Table 1). Since S-VSP{alpha} in transgenic tobacco plants is found solely in the PBS soluble fraction (Guanoune et al., 1999), which, in turn, represents about 70–80% of the total protein content (data not shown), it was assumed that the effect of S-VSP{alpha} accumulation on total lysine content is about 70–80% of that found in the soluble fraction.


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  		Table 1. Mean values of total PBS soluble lysine in control (WT) and several transgenic tobacco linesValues represent the free lysine pool and protein-bound lysine in PBS soluble proteins.
 
The level of vacuolar S-VSP{alpha} in transgenic tobacco plants doubled in homozygous compared to heterozygous plants (Guenoune et al., 2002). Therefore, a similar elevation in the level of both vacuolar and chloroplast S-VSP{alpha} transgene proteins in the homozygous line CE20/W2 would be expected. This should bring S-VSP{alpha} to a level of 12% of total soluble protein and increase total soluble lysine up to 30%. In the present work, additional evidence is provided that chloroplasts can accumulate alien proteins to high level. Moreover, it has been shown that directing a transgene protein to more than one organelle in a single plant is a feasible approach that can lead to further accumulation of a candidate protein. By applying it to other transgene proteins, this approach may be utilized to improve plant nutritional quality as well as to increase the benefit of using plants as bioreactors.


   Acknowledgements
 
The authors wish to thank Mr Yigal Avivi for editing the manuscript. This project was supported by the Chief Scientist of the Ministry of Agriculture, grant No. 259-0088 and is Contribution No. 139 (2001 series) from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel.


   References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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