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First published online February 28, 2002; 10.1104/pp.010615 Plant Physiol, April 2002, Vol. 128, pp. 1282-1290
Constitutive Expression of the
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Polyhydroxybutyrate (PHB) is a member of a class of
thermoelastic polymers called polyhydroxyalkanoates that serve many
bacteria as intracellular storage molecules for carbon and energy.
Transgenic plants provide a potential means of producing this polymer
cost-effectively. To date, however, few reports of the successful
production of this polymer have been published, with the exception of
work with transgenic Arabidopsis. Using a variety of chimeric
constructs, we have determined that the constitutive,
chloroplast-localized expression of one of the genes involved in PHB
production
the 
-ketothiolase (phbA) geneis
detrimental to the efficient production of transgenic PHB. The
alternate use of either inducible or somatically activated promoters
allowed the construction of transgenic PHB-producing potato
(Solanum tuberosum) and tobacco (Nicotiana
tabacum) plants, although the amount of PHB formed was still
rather low. Taking advantage of an inducible promoter, the maximal
amount of PHB produced in transgenic potato was 0.09 mg
g
1 dry weight. In transgenic tobacco using a somatically
activated promoter, up to 3.2 mg g1 dry weight was
accumulated. In Arabidopsis, the formation of high levels of PHB had
previously been shown to be accompanied by severe negative effects on
growth and development of the plant. Phasins are proteins known from
PHB-producing bacteria speculated to serve as protectants against the
highly hydrophobic surface of the PHB granules in the bacterial
intracellular milieu. Co-expression of the phasin gene in parallel with
the PHB synthesis genes, however, did not lead to reduced symptom development.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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The use of transgenic plants for the
production of novel compounds represents one important aspect of plant
biotechnology. Whereas multiple examples exist with respect to the
production of novel compounds to be used in food and feed, much less
work has been done with respect to the use of transgenic
plants for production of compounds for non-food purposes.
Polyhydroxyalkanoates (PHAs) are polymers that serve many bacteria as
intracellular storage molecules for carbon and energy. These molecules
occur alone or together with triacylglycerols (Alvarez et al., 2000
). The homopolymer poly(3-hydroxybutyrate) (PHB) is the most common type
of PHA, although many other PHAs are known (Steinbüchel and
Valentin, 1995). Starting from acetyl-CoA, PHB is
synthesized in bacteria by the consecutive action of three genes:
-ketothiolase (phbA), acetoacetyl CoA reductase
(phbB), and PHB synthase (phbC). Thermoelastic
polymers such as PHB have a number of advantages over polymers derived
from oil: They are biodegradable, their production is essentially
neutral with respect to carbon dioxide balance, and they are
biologically renewable and are, therefore, in line with the goal of
sustainable development (Steinbüchel and Füchtenbusch,
1998).
The use of recombinant micro-organisms to efficiently produce PHB and
PHAs has been described extensively. The main disadvantage of this
production route is its high cost (Choi and Lee, 1999). Plants
obviously represent an attractive alternative for PHB production. Pioneering work performed by Poirier et al. (1992) in Arabidopsis showed that PHB synthesis is feasible in plants. A dramatic increase in
the amount of PHB produced by Arabidopsis plants was achieved when PHB
synthesis was engineered in chloroplasts (Nawrath et al., 1994).
However, Arabidopsis obviously will not be the crop used for PHB
production. Surprisingly, few reports of PHB production in crop
plants have appeared, and those that have reported only very low
amounts of PHB being produced (compare with e.g. John and Keller, 1996;
Nakashita et al., 1999). The only exception is the work resulting in
transgenic Brassica napus plants with seeds expressing up to
7% (w/v) PHB by weight (Valentin et al., 1999; Houmiel et al.,
1999).
We recently reported the efficient production of PHB in Arabidopsis
leaf chloroplasts where leaf levels up to 40% of dry weight were
reached, thus, confirming and extending earlier observations by Nawrath
et al. (1994). These high amounts were, however, accompanied by severe
growth reduction of the plant and the development of chlorotic leaves.
We, therefore, started a series of experiments with essentially two goals: analysis of plants other than Arabidopsis (notably potato [Solanum tuberosum] and tobacco [Nicotiana tabacum]) for their suitability as hosts for efficient PHB production; and analysis of strategies to prevent the negative effects of PHB synthesis on growth and development.
Evidence will be described suggesting that the constitutive expression of one of the PHB synthesis genes (phbA) is detrimental to plant growth as early as during the transformation step. When put either under an inducible or a somatically activated promoter, transgenic tobacco and potato plants were obtained that expressed all three PHB genes. However, the amount of PHB formed was nevertheless low, suggesting that, in this case, what is true for Arabidopsis is not necessarily true for potato and tobacco. Results obtained following two strategies trying to circumvent the negative effects of PHB accumulation on growth and development in Arabidopsis will also be described.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Transformation of Tobacco and Potato with Chimeric Gene Constructs Containing the Three PHB Genes under a Constitutive Promoter Leads to a Low Number of Transgenic Plants, Only a Few of Which Produce Minor Amounts of PHB
The construct pBI ABC, which contains the three PHB-synthesis
genes fused to a plastid-targeting signal under the control of the
constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter, was used
for Agrobacterium tumefaciens-mediated transformation experiments using leaf discs of tobacco and potato. In tobacco, a much
reduced transformation frequency was observed yielding only 47 lines in
total from several transformation batches, which represents about
one-tenth of the number of lines expected when compared with other
transformations. In potato, the situation was even worse. Of five
transformation experiments, which normally would have yielded several
hundred transformants, no transgenic lines were obtained at all. The
transgenic tobacco lines grown in tissue culture were directly screened
for their 3-hydroxybutyrate content via gas chromatography-mass
spectroscopy (GC-MS) analysis. PHB accumulation was observed in only
three lines, the maximal amount being 91 µg 3-hydroxybutyrate
g1 fresh weight (Table
I). After transfer to the greenhouse,
this already low level fell even further (< 5 µg 3-hydroxybutyrate g1 fresh weight). These results were most
surprising given the fact that the identical construct resulted in
transgenic Arabidopsis plants containing up to 40% of their dry weight
as PHB (Bohmert et al., 2000).
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Expressing Chimeric Genes Bearing the -Ketothiolase Gene
(phbA) under the Control of a Constitutive Promoter Leads
to a Reduced Transformation Efficiency
One possible explanation for the low efficiency of transformation
described above is that one of the three chimeric genes directing PHB
synthesis is specifically detrimental for plant growth and development
during the transformation procedure. To investigate this hypothesis and
possibly to identify the gene responsible for this effect, we performed
a series of transformation experiments where, in each case, only one of
the three genes (constructs pBI-TP-Thio, pBI-TP-Red, and pBI-TP-Syn;
Nawrath et al., 1994) was used. Whereas transformation of either
tobacco or potato using the construct pBI-TP-Syn led to normal
transformation efficiencies and the use of the construct encoding the
acetoacetyl-CoA reductase led to only a slightly decreased
transformation efficiency, the use of the construct pBI-TP-Thio
encoding the -ketothiolase led to a drastically reduced
transformation efficiency in case of both tobacco and potato (Fig.
1).
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To determine whether this negative influence of the acetoacetyl-CoA
reductase gene on transformation frequency was also true in
Arabidopsis, we analyzed the efficiency of transformation using all
three genes singly and in various combinations. The single constructs
pBI-TP-Red and pBI-TP-Syn and a chimeric construct containing both the
PHB synthase and the acetoacetyl-CoA reductase gene led to normal
transformation efficiencies (between 1% and 3% of the seeds being
transformed). In strong contrast to this, the rate dropped down to
0.01% whenever the -ketothiolase gene was contained in the
construct either alone (pBI-TP-Thio) or in combination with the
acetoacetyl-CoA reductase gene or the acetoacetyl-CoA reductase plus
the PHB synthase gene (data not shown).
These experiments, thus, strongly suggest that in all three plant
species analyzed, i.e. tobacco, potato, and Arabidopsis, the expression
of the -ketothiolase gene has detrimental effects on transformation
efficiency, thus, preventing the analysis of these plant species with
respect to their suitability to serve as hosts for PHB production.
Construction of Chimeric Genes Allowing the Inducible or Somatically Activated Expression of the Thiolase Gene
The data described above suggest the expression of the
phbA gene during the transformation procedure is responsible
for the reduced transformation efficiency. We, therefore, followed two strategies to alter the expression of phbA. In the first, we
used the prp-1 promoter from potato. The prp-1
promoter was originally identified as a pathogen-inducible promoter
(Martini et al., 1993), but it can also be induced via external
application of salicylic acid. Inducing the expression of
phbA after transgenic plant regeneration should prevent any
potentially deleterious effects of phbA expression during
the transformation and regeneration. In the second approach, we took
advantage of the maize transposable element Ac. The
Ac element was inserted between the CaMV 35S promoter and
the coding region of the -ketothiolase gene, thus, allowing
expression of the -ketothiolase gene only after the mobile element
has been excised due to the establishment of the link between the
promoter and the coding sequence. Frequent somatic excision of Ac has
been reported in tobacco, potato, and Arabidopsis (Van Sluys et al., 1987; Knapp et al., 1988; Taylor et al., 1989; Keller et al., 1993;
Lawson et al., 1994).
Both constructs (pBinARHygprpABC for inducible expression of phbA and pBinARHygacABC bearing the transposable element; see Fig. 2) also contain the acetoacetyl-CoA reductase gene (phbB) and the PHB synthase gene (phbC), both of which are constitutively expressed under the control of the CaMV 35S promoter. All proteins are targeted to the plastid.
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Alternative Constructs Lead to a Significant Increase in Transformation Efficiency for Both Tobacco and Potato
A. tumefaciens-mediated transformation of tobacco with
the construct pBinARHygprpABC led to 66 transgenic tobacco
prpABC lines. All lines were grown in the greenhouse and
sprayed with 5 mM salicylic acid once a day for a
period of 15 weeks. Leaf samples were taken from the edge of the top
one-third of mature leaves, which is the oldest part of the leaf
(analysis of the distribution of PHB throughout the whole leaf revealed
that this part accumulates the highest amount of PHB; data not shown).
Samples were taken 10 d before the beginning of the induction with
salicylic acid and 4, 22, 44, and 88 d after the 1st d of
induction. PHB accumulation was shown to take place in 23 of the 66 transgenic tobacco prpABC lines screened, the maximal amount
of PHB observed being 50 µg 3-hydroxybutyrate
g1 fresh weight (corresponding to 420 µg
3-hydroxybutyrate g1 dry weight [Table I]),
detected 32 d after onset of induction. Further analysis showed
that the promoter was leaky in some of the transgenics. No significant
changes in phenotype were observed.
Using the construct pBinARHygacABC, 80 transgenic tobacco
acABC lines were obtained. PHB accumulation was detected in 21 of the 80 plants analyzed with a maximal production of 375 µg
3-hydroxybutyrate g1 fresh weight,
corresponding to 3.2 mg 3-hydroxybutyrate g1
dry weight (Table I). Some of the lines displayed phenotypic changes
such as chlorosis, changes in leaf morphology or increased number of
trichomes. These changes could not be correlated, however, with the
amounts of PHB produced.
Both constructs were also used for A. tumefaciens-mediated
transformation of potato. Transformation using the construct
pBinARHygprpABC led to 24 transgenic potato prpABC lines. Ten
transgenic potato prpABC lines accumulated some PHB, the
maximal level after induction being 11 µg 3-hydroxybutyrate
g1 fresh weight (corresponding to 87 µg
3-hydroxybutyrate g1 dry weight; Table I).
Analysis of negative controls, which were not sprayed, showed that the
promoter was leaky in potato, too (Fig.
3).
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Using the construct pBinARHygacABC for potato transformation, 13 transformants were generated, of which two lines produced some PHB
(maximal 2 µg 3-hydroxybutyrate g1 fresh
weight; Table I). In addition to leaf extracts, tubers of all
transgenic potato lines were also screened for PHB accumulation. They
contained detectable but very low amounts of PHB (< 1 µg 3-hydroxybutyrate g1 fresh weight). Neither the
prpABC transgenics nor the acABC lines displayed outward phenotypic changes.
Creation and PHB Analysis of Transgenic acABC and prpABC Lines in Arabidopsis
Both constructs, pBinARHygprpABC and pBinARHygacABC, were also
used to transform Arabidopsis. This was done to check the functionality of the constructs in a known system on one hand, and on the other hand,
to see whether the use of these different expression systems may allow
the negative effects on growth observed earlier to be overcome (Bohmert
et al., 2000).
In the pBinARHygprpABC construct, 151 transgenic prpABC lines were obtained. After 3 weeks, the Arabidopsis plants had developed a rosette and started to bolt. Leaf samples were taken from all plants before onset of induction with salicylic acid and 18 d after induction.
Most of the transgenic Arabidopsis prpABC lines produced
PHB before the first treatment with salicylic acid. The plant that accumulated the highest amount of PHB before spraying (2.7 mg 3-hydroxybutyrate g1 fresh weight) showed
severe growth reduction (Fig. 4).
The same plant produced the maximal amount of PHB measured
among all Arabidopsis prpABC lines after 18 d of spraying (11 mg 3-hydroxybutyrate g1 fresh weight; Table
I). Other plants accumulated up to 6 mg 3-hydroxybutyrate
g1 fresh weight after the induction period and
displayed no negative effects on their growth.
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Transformation of Arabidopsis with the construct pBinARHygacABC
generated 80 transgenic acABC lines. The maximal amount of PHB observed
was 1.4 mg 3-hydroxybutyrate g1 fresh weight
(Table I).
Co-Expression of the Phasin Gene in PHB-Producing Arabidopsis Plants Does Not Alleviate the Negative Effects on Growth and Development
Phasins are proteins expressed in PHA-producing bacteria that are
associated with the surface of the PHA granules. Bacteria devoid of
phasins produce less PHB and, furthermore, display a slightly reduced
growth rate. Phasin production has, therefore, been suggested to be a
protective mechanism against the highly hydrophobic PHB granule
(Steinbüchel et al., 1995). To test whether phasins may exert
such a protective effect in plants, we decided to express phasins in
transgenic Arabidopsis plants in the presence or absence of PHB
synthesis genes.
To test the effects of phasin proteins alone, Arabidopsis was transformed with the construct pBI P encoding a phasin protein fused to a chloroplast transit peptide and driven by the constitutive CaMV 35S promoter (Fig. 2). Seventy-two transgenic lines were analyzed, none of which showed any alteration in outward phenotype. RNA and protein-blot analysis substantiated the expression of the phasin gene (data not shown).
In a next step, the construct pBinARHygABC P, containing the three
genes needed for PHB synthesis and the phasin gene (all under the
control of the CaMV 35S promoter and all with a chloroplast-targeting sequence), was used (Fig. 2). In total, 166 transgenic Arabidopsis lines were generated and analyzed for PHB production via GC-MS analysis. The maximal amount of PHB measured in leaf samples was 4.9 mg
3-hydroxybutyrate g1 fresh weight
(Table I). High PHB-producing lines showed reduced growth and a chlorotic pattern in their leaves. The highest amount of
PHB produced in transgenic lines without phenotypic changes was 1.5 mg
3-hydroxybutyrate g1 fresh weight.
Transmission electron microscopy of leaf samples of PHB-producing ABC P
lines revealed that the polymer accumulated in granules located in the
stroma of the plastids. In comparison with former results from
transgenic lines with constitutive expression of the three
phb genes without the phasin gene (Bohmert et al., 2000), no
difference in granule size was observed. Preliminary results from
immunolocalization of the phasin protein suggest predominant localization of the phasin protein at the surface of the PHB granules (data not shown). The signal density obtained for the transgenic lines
was comparable with those in R. eutropha controls (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Despite the impressive, large amounts of PHB produced using
Arabidopsis as host plant for PHB expression and despite the obvious economic potential of plants as factories for PHB production, there is
only one report in the literature of a crop plant producing significant
levels of PHB (in seeds of B. napus; Valentin et al., 1999;
Houmiel et al., 1999). The data described here enabled us to identify
one critical parameter with respect to PHB production in plants: the
expression of the -ketothiolase gene, phbA. Results obtained with a variety of chimeric constructs used for transformation of tobacco, potato, and Arabidopsis indicate that the constitutive expression of the -ketothiolase gene is responsible for the
drastically reduced transformation efficiency observed. To test this
hypothesis, two chimeric genes were used where either the expression of
phbA was put under the control of an inducible promoter or
the coding sequence of phbA was separated from the promoter
by the presence of a transposable element, thus, leading only to
somatic activation of expression. In both cases, generation of
transgenic lines was possible in all three plant species.
These data suggest that preventing the expression of phbA
during the transformation/regeneration procedure is sufficient to allow
generation of transgenic plants. The toxic effects exerted by
phbA expression could result from PHB biosynthesis
intermediates (acetoacetyl-CoA and 3-hydroxybutyryl-CoA) or derivatives
of these intermediates, the depletion of the acetyl CoA pool, or
interactions of the -ketothiolase with other proteins or substrates.
One candidate interfering substance, acetoacetyl-CoA, is present in the
cytosol, where it is an intermediate of the mevalonate pathway leading
to isopentenyl diphosphate, the central precursor for all isoprenoids.
In the plastid, isopentenyl diphosphate proceeds via the dioxyxylulose
5-phosphate pathway (Arigoni et al., 1997; Lichtenthaler et al., 1997;
Lange et al., 2000). Therefore, acetoacetyl-CoA is not expected to be
present in the plastid. Another observation supports this assumption:
Measurements of transgenic Arabidopsis, tobacco, and potato lines with
constitutive expression of phbB and phbC targeted
to the plastids did not reveal any PHB accumulation (data not shown).
Because acetoacetyl-CoA is quite similar to -ketoacyl ACP, one may
speculate about an interference of acetoacetyl-CoA with the reaction of
-ketoacyl ACP synthase (KASIII) in fatty acid biosynthesis. To
further analyze this possibility, fatty acids were determined in
PHB-producing representatives of the lines ABC, prpABC, and ABC P. None
of the analyzed lines displayed changes in total fatty acid levels or
in fatty acid composition (data not shown), which is in agreement with
similar data obtained for PHB-producing Brassica napus seeds
(Houmiel et al., 1999). Therefore, these data do not support an
interference of acetoacetyl-CoA with the reaction catalyzed by KASIII.
The second aim of this work was to test possible strategies to prevent
the severe impairment in growth and development observed in Arabidopsis
plants accumulating high amounts of PHB (
3 mg g1 fresh weight; Bohmert et al., 2000).
Essentially two strategies were followed. First, using an inducible
promoter system transgenic prpABC, Arabidopsis lines were grown until
they developed a fully expanded rosette and were subsequently treated
with salicylic acid to induce phbA gene expression. This
procedure led to transgenic lines accumulating 6 mg 3-hydroxybutyrate
g1 fresh weight without any alteration in
phenotype. This is twice the amount observed in phenotypically
comparable Arabidopsis lines constitutively expressing the
phbA gene (Bohmert et al., 2000). Transgenic lines bearing
the inducible promoter produced up to 11 mg PHB
g1 fresh weight. This is a fairly high value
considering that full phbA gene expression was present for
only 18 d. This line, however, displayed growth retardation.
The second strategy for overcoming growth reduction in PHB-accumulating Arabidopsis lines was directed toward possible negative effects caused by the PHB itself. One could imagine that PHB granules may interfere with plastidial structures and, thereby, hinder their function. In PHB-accumulating bacteria, PHB granules are associated with proteins called phasins. One could speculate that the phasins prevent interference of PHA granules with other structures such as thylakoid membranes. To test possible positive effects of phasins on PHB accumulation in transgenic plants, the phasin gene phaP of R. eutropha was constitutively expressed in Arabidopsis. Expression of the phaP gene alone did not cause any outward phenotypic changes. Expression of the three phb genes in conjunction with phaP led to PHB production in transgenic lines; however, higher PHB concentrations were again accompanied by growth reduction. Therefore, phaP gene expression does not mask or prevent the deleterious effects of PHB expression.
In conclusion, the data presented suggest that constitutive expression
of -ketothiolase leads to problems during transformation of
different plant species using an A. tumefaciens-mediated in planta transformation procedure (Arabidopsis) and tissue culture transformation procedures (tobacco and potato). Strategies that overcome this problem include the use of an inducible promoter system
or a somatically activated expression system. Unfortunately, the
association of growth impairment with high PHB production could not be
overcome using these strategies.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Plasmid Constructions
A 439-bp BamHI fragment of the plasmid pBS42-1
(Martini et al., 1993) was ligated in sense orientation into the
BamHI restriction site of plasmid pBS-SK (Stratagene, La
Jolla, CA). The resulting plasmid was digested with the restriction
enzymes SmaI and NotI, and the
overhanging ends were filled by T4 DNA polymerase. This 462-bp fragment
containing the prp1-1 promoter of potato (Solanum tuberosum; Martini et al., 1993) was ligated into the plasmid pBI-TP-Thio (Nawrath et al., 1994), which had been digested with the
restriction enzymes HindIII and XbaI and
which had the 5' ends filled-in, resulting in plasmid pBIprpA.
Plasmid pBI-TP-Syn (Nawrath et al., 1994) was digested with the
restriction enzymes EcoRI and XbaI and
had the 5' ends filled-in. This 2.1-kb fragment was ligated in sense
orientation into the SmaI site of plasmid pBinARHyg
(Becker, 1990). The resulting plasmid, pBinARHygC, was linearized with
EcoRI, and the 5' ends were filled-in. A
HindIII/EcoRI digestion of plasmid
pBI-TP-Red (Nawrath et al., 1994) generated a 2.1-kb fragment ligated
in sense orientation into the linearized plasmid pBinARHygC. The
resulting plasmid, pBinARHygBC, was linearized with
HindIII, and the 5' ends were filled-in. An
XbaI/EcoRI digestion of the plasmid
pBIprpA generated a 2.3-kb fragment that was ligated in sense
orientation into the linearized plasmid pBinARHygBC to generate the
construct pBinARHygprpABC. This construct contained three
cassettes, each of them bearing one of the three phb
genes with a plastidial-targeting sequence, a poly(A) adenylation
sequence, and either the CaMV 35S promoter (phbB and
phbC) or the inducible prp1-1 promoter
(phbA).
The constitutive CaMV 35S promoter of plasmid pBI121 (CLONTECH, Palo
Alto, CA) was amplified by PCR using the 37-mer oligonucleotide 5'-ggT
CTA gAA CTA gTg TCC CCC gTg TTC TCT CCA AAT g-3' complementary to the
promoter's 5' end in combination with the 37-mer oligonucleotide 5'-gCg CAA gCT TgC ATg CCT gCA ggT CCC CAg ATT AgC C-3' (PHB-2) to
generate a SpeI site upstream of the XbaI
site at the promoter's 3' end. After digestion of the 800-bp PCR
fragment with the restriction enzymes XbaI and
HindIII, the promoter was ligated together with a 1.8-kb
XbaI/EcoRI fragment of plasmid
pBI-TP-Thio (Nawrath et al., 1994) into the
HindIII/EcoRI site of plasmid pBS-SK
(Stratagene), resulting in plasmid pSKASpe. Plasmid pBinARHygBC was
digested with the restriction enzyme HindIII, and the 5'
ends were filled-in. A HindIII/EcoRI
digestion of plasmid pSKASpe led to a 2.6-kb fragment that was ligated
in sense orientation into the linearized plasmid pBinARHygBC to
generate the construct pBinARHygSpeABC.
The oligonucleotides 5'-ggA CTA gTC Agg gAT gAA AgT Agg ATg-3' (PHB-3)
27-mer and 5'-ggA CTA gTA ggg ATg AAA Acg gTC gg-3' (PHB-4) were used
to amplify the transposable Ac element from maize from
the plasmid pT12 (T. Altmann, unpublished data; Müller-Neumann et al., 1984; Pohlmann et al., 1984) adding SpeI
restriction sites at both ends. The 4.5-kb PCR-product was digested
with SpeI and ligated in sense orientation into the
vector pBinARHygSpeABC linearized with SpeI,
resulting in plasmid pBinARHygacABC. This plasmid contains three
cassettes, each of them bearing one of the three phb
genes with a plastidial-targeting sequence, a poly(A) adenylation
sequence, and the CaMV 35S promoter. The transposable Ac
element of maize was located upstream of the phbA gene
and its plastidial-targeting sequence.
The plasmid pSKSaE17 bearing a 1,692-bp subfragment of E5000 (Wieczorek
et al., 1995) was used as a template to amplify the 579-bp coding
region of the phaP gene of R. eutropha
(Hanley et al., 1999). Using the 30-mer oligonucleotides 5'-AAT CCC ggg
TgA TCC TCA CCC Cgg AAC AAg-3' (PHB-7) and 5'-ggg gAg CTC TTC AAC gCA
ggC AgT TAT CAg-3' (PHB-8) led to the introduction of a
SmaI restriction site at the 5' end and a
SacI restriction site at the 3' end. The resulting PCR
product was cut with SmaI and SacI and
was ligated into the SmaI/SalI site of
the vector pBI-TP-Red (Nawrath et al., 1994). The resulting construct,
pBI P, harbored phaP under control of the CaMV 35S
promoter and targeted to the plastid.
The constitutive CaMV 35S promoter of the plasmid pBinARHyg (Becker,
1990) was amplified by PCR using the 40-mer oligonucleotide 5'-gCg CAA
gCT TAC TAg Tgg ATC CgC Atg CCT gCA ggT CCC C-3' (PHB-10) and the
39-mer oligonucleotide 5'-ggT CTA gAg TCC CCC gTg TTC TCT CCA AAT gAA
ATg AAC-3' (PHB-9). After digestion of the 800-bp PCR fragment with the
restriction enzymes HindIII and XbaI, the promoter was ligated to the
HindIII/XbaI site of the plasmid
pBI-TP-Thio (Nawrath et al., 1994). The 2.4-kb
HindIII/EcoRI fragment of the resulting
plasmid was introduced into the HindIII site of the plasmid pBinARHygBC to create the plasmid pBinARHyg2000SpeABC. The
1.9-kb HindIII/EcoRI fragment of plasmid
pBI P was blunt-end-ligated to the SpeI site of
pBinARHyg2000SpeABC. The resulting plasmid, pBinARHygABC P, contained
four cassettes, each of them bearing one of the three
phb genes or the phasin gene. Every gene contained a
plastidial-targeting sequence, the CaMV 35S promoter, and a poly(A)
adenylation sequence.
Construction of the plasmids pBI AB, pBI ABC, pBI-TP-Thio, pBI-TP-Red,
and pBI-TP-Syn has been described previously (Nawrath et al., 1994;
Bohmert et al., 2000).
Plant Material and Transformation
The plasmids pBI-TP-Thio, pBI-TP-Red, pBI-TP-Syn,
pBinARHygprpABC, pBinARHygacABC, pBI P, and pBinARHygABC P were
electroporated into Agrobacterium tumefaciens C58C1
harboring plasmid pMP90 and used to transform Arabidopsis C24 plants
via dipping (Clough and Bent, 1998). Transformed seeds were selected on
Murashige and Skoog medium (Murashige and Skoog, 1962; DUCHEFA,
Haarlem, The Netherlands) containing 1% (w/v) Suc and 100 µg
mL1 kanamycin or 20 µg mL1 hygromycin.
Plants were grown at 20°C with a 16-h-light, 8-h-dark regime with
approximately 250 µmol photons m2
s1.
Potato cv Desiree was obtained from Saatzucht Lange AG (Bad Schwartau,
Germany). Transformation of potato was performed using the protocol of
Rocha-Sosa et al. (1989). Depending on the construct used, transgenic
plants were selected on kanamycin- or hygromycin-containing medium
(Dietze et al., 1995). Plants were maintained in tissue culture with a
16-h-light, 8-h-dark regime on Murashige and Skoog medium (Murashige
and Skoog, 1962; DUCHEFA) containing 2% (w/v) Suc. In the
greenhouse, plants were grown under the same light regime with a
minimum of 250 µmol photons m2 s1 at
22°C.
Seeds of tobacco (Nicotiana tabacum L. cv Samsun NN)
were obtained from Vereinigte Saatzuchten (Ebstorf, Germany). Tobacco transformation using A. tumefaciens-mediated gene
transfer was carried out as described previously (Rosahl et al., 1987;
Komari, 1989). Plants in tissue culture were grown with a 16-h-light, 8-h-dark regime on Murashige and Skoog medium (Murashige and Skoog, 1962; DUCHEFA) containing 2% (w/v) Suc. In the greenhouse,
plants were grown under the same light regime with a minimum of 250 µmol photons m2 s1 at 25°C during the
light period and 20°C during the dark period.
Treatment of Transgenic prpABC Lines with Salicylic Acid
All experiments with transgenic prpABC lines were performed in the greenhouse. The plants were sprayed once a day. Samples were taken from mature leaves. Transgenic prpABC lines of tobacco were sprayed with 5 mM salicylic acid for a period of 15 weeks. Leaf samples were taken from the edge of the top one-third of mature leaves. Sampling was performed 10 d before induction with salicylic acid and 4, 22, 44, and 88 d after the 1st d of induction.
Transgenic prpABC lines of potato were sprayed with 1 mM salicylic acid for a period of 5 weeks. Leaf samples were taken the day before the beginning of the induction with salicylic acid, the day after the first spraying, and 11 and 33 d after the first induction.
First spraying of transgenic prpABC lines of Arabidopsis with 1 mM salicylic acid was performed on plants beginning to bolt (4 weeks after germination) and was continued for a period of 18 d. Leaf samples were taken before the first salicylic acid induction and at the end of the spraying period.
Transgenic prpABC lines of B. napus were sprayed with 1 mM salicylic acid during 6 weeks. Leaf samples were taken from the edge of the top one-third of mature leaves. The first samples were taken before induction with salicylic acid. The next were taken the next day, after which sampling was continued weekly.
Analysis of PHB by GC-MS
Extraction and derivatization of 20 to 250 mg of leaf material
or 100 mg of tuber material was performed as described previously (Bohmert et al., 2000). An aliquot (1 µL) of the derivative was injected into a GC-MS system (AS 2000 autosampler, GC 8000 gas chromatograph and a MD 800 quadrupole mass spectrometer; all
ThermoQuest, Manchester, UK) using a split ratio of 25:1. Single-ion
monitoring was used to detect the m/z 189, which
corresponds to the major fragment of 3-trimethylsilyl oxybutanoic acid
ethyl ester after 3,4 cleavage. Chromatography was carried out using a
30-m × 250-µm DB 5MS column (J&W Scientific, Folsom, CA).
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ACKNOWLEDGMENT |
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We thank Megan McKenzie (Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm/Potsdam, Germany) for critical reading of the manuscript.
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FOOTNOTES |
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Received July 10, 2001; returned for revision August 30, 2001; accepted January 19, 2002.
1 This work was supported by the Bundesministerium für Landwirtschaft (to K.B. and I.B.) and by the German Agricultural Ministry.
2 Present address: Metabolix Inc., 303 Third Street, Cambridge, MA 02142-1126.
* Corresponding author; e-mail bohmert{at}metabolix.com; fax 617-492-1996.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010615.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus.
Planta
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-Ketothiolase Gene in
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Polyhydroxybutyrate-Producing Plants
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