Journal of Experimental Botany, Vol. 53, No. 376, pp. 1867±1870, September 2002
DOI: 10.1093/jxb/erf046
Combined expression of S-VSPa in two different
organelles enhances its accumulation and total lysine
production in leaves of transgenic tobacco plants
Dana Guenoune1, Rachel Amir2, Hanna Badani1, Shmuel Wolf3 and Shmuel Galili1,4
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
Received 11 January 2002; Accepted 3 June 2002
Abstract
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-VSPa to two different organellesÐchloroplasts
and vacuolesÐin a single transgenic plant signi®cantly increased its accumulation. Accumulation of
S-VSPa 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: Chloroplasts, gene expression, improved
nutritional quality, soybean vegetative storage proteins, total
lysine content, transgenic tobacco plants.
Introduction
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 aminoacid-rich seed storage proteins accumulate to high levels
when expressed in seed tissues, resulting in a signi®cant
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
cauli¯ower mosaic virus (CaMV) 35S promoter resulted in
only a minor success (Galili et al., 2002). These proteins,
4
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). SVSPs contain about 7% lysine and accumulate to high
levels inside vacuoles of vegetative tissues of both wildtype (WT) soybean (Staswick, 1994) and transgenic
tobacco plants (Guenoune et al., 1999). Furthermore, as
the accumulation of S-VSPa 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
Plant material
The plant material used in this study consisted of Nicotiana tabacum
L. cv. Samsun NN, soybean (Glycine max Merr.), and the following
To whom correspondence should be addressed. Fax: +972 3 9669642. E-mail: [email protected]
ã Society for Experimental Biology 2002
1868 Guenoune et al.
transgenic tobacco plants: line CE20, expressing S-VSPa inside the
chloroplasts and line W2, expressing S-VSPa 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-VSPa to chloroplasts, the S-VSPa encoding
DNA sequence of the plasmid pKSH1 (Mason et al., 1988) was
ampli®ed by polymerase chain reaction (PCR) using PWO DNA
polymerase (Boehringer) to form an in-frame SphI site. This site
substituted the ®rst alanine codon GCC of the mature S-VSPa
protein (Mason et al., 1988) by a methionine translation initiation
codon ATG. The primers used were 5¢-AGCATGCGTACTCCGGAGGTGAAATGC-3¢ and 5¢-AGGAACTACTGAATGTAGTACAG-3¢. The ampli®ed 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 W 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-VSPa 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 (SDSPAGE), Western blot analysis, Coomassie Brilliant Blue staining,
and quantitative estimation of the S-VSPa 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 mg were used to analyse the amino
acid composition as previously described (Karchi et al., 1993).
Results and discussion
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 in¯uenced by their subcellular location
Fig. 1. Effect of leaf age on accumulation of S-VSPa in transgenic
tobacco plants. Leaf protein extracts (20 mg) were separated on SDS±
PAGE and analysed by Western blot using anti S-VSPs antibodies.
(A) T0 plant of line CE20 expressing S-VSPa inside its chloroplasts.
(B) F1 plant derived from the cross between transgenic tobacco plants
of line CE20 and line W2 (expressing S-VSPa 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.
(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-VSPa 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-VSPa 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.
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-VSPa 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-VSPa 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
S-VSPa expression in two organelles 1869
2.3% in leaf No. 18 (Fig. 2, line CE20). The level of
S-VSPa relative to the total soluble leaf proteins in line
W2, in which S-VSPa 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-VSPa in line CE20 was slightly, but
not signi®cantly, 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 signi®cantly higher
compared to its expression inside the vacuoles, suggesting
that the chloroplast may be an additional candidate storage
organelle for nuclear encoded transgene proteins.
Although chloroplasts are suitable storage organelles for
S-VSPa accumulation, the total S-VSPa was not higher
Fig. 2. Relative levels of S-VSPa in leaves of different age in several
transgenic tobacco lines. Protein extracts (20 mg) were separated on
SDS-PAGE and stained with Coomassie Brilliant Blue, followed by
scanning the stained gels. W2, line expressing S-VSPa inside the
vacuoles; CE20, line expressing S-VSPa inside the chloroplasts;
CE20/W2, F1 plants derived from the cross between lines CE20 and
W2. S-VSPa level in line CE20/W2 is represented by stacked bars in
which the lower and upper bars represent the relative levels of
S-VSPa accumulated inside the vacuoles and the chloroplasts,
respectively. The level of S-VSPa 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 (6SE).
than the level obtained when this protein was targeted to
the vacuoles. It was tested, therefore, whether directing a
transgene protein, such as S-VSPa, 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-VSPa transgene proteins accumulated in both organelles
(Fig. 1B). The upper, 27 kDa band, which co-migrates with
the S-VSPa of soybean, corresponds to the vacuolar
S-VSPa protein and the lower, 24 kDa band, corresponds
to the chloroplast S-VSPa protein. Both S-VSPa 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-VSPa in these
F1 plants is shown in Fig. 2 (line CE20/W2). The difference
in the molecular weight between the two S-VSPa
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-VSPa in the F1 line CE20/W2 was similar to the
sum of the S-VSPa levels obtained in lines CE20 and W2
(Fig. 2). In leaf No. 18 of line CE20/W2, however,
signi®cantly higher S-VSPa levels were obtained compared to the sum of the S-VSPa levels obtained in lines
CE20 and W2. The results indicate that the accumulation
of S-VSPa in one organelle is not affected by its
accumulation in an additional organelle. The accumulation
of S-VSPa 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
signi®cantly 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-VSPa 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-VSPa accumulation on total lysine content is
about 70±80% of that found in the soluble fraction.
Table 1. Mean values of total PBS soluble lysine in control (WT) and several transgenic tobacco lines
Values represent the free lysine pool and protein-bound lysine in PBS soluble proteins.
Lines
Total soluble lysinea
mol%b
WT (control untransformed plants)
W2Ht (heterozygous for S-VSPa)
CE20 (heterozygous for S-VSPa modi®ed to accumulate inside the chloroplasts)
CE20/W2 (F1 plants of the cross CE203W2)
a
b
c
Values represent soluble lysine plus lysine content in soluble proteins.
Values represent means 6SE from three plants per line.
Different letters designate signi®cantly different values at P <5%, as measured by means, F-test.
4.8660.05
5.3360.07
5.3660.02
5.7 60.19
% of Control
c
c
b
ab
a
100
107
110
117
1870 Guenoune et al.
The level of vacuolar S-VSPa 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-VSPa transgene proteins in the homozygous line CE20/
W2 would be expected. This should bring S-VSPa 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 bene®t 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.
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