Genetic Transformation and Hybridization
Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): Significant contents of polyhydroxybutyrate are associated with growth reduction
Received: 16 August 2002 Revised: 31 January 2003 Accepted: 14 February 2003
Abstract The pathway for synthesis of polyhydroxybutyrate (PHB), a polyester produced by three bacterial enzymes, was transferred to the tobacco plastid genome by the biolistic transformation method. The polycistronic phb operon encoding this biosynthetic pathway was cloned into plastome transformation vectors. Following selection and regeneration, the content and structure of plant-produced hydroxybutyrate was analysed by gas chromatography. Significant PHB synthesis was limited to the early stages of in vitro culture. Within the transformants, PHB synthesis levels were highly variable. In the early regeneration stage, single regenerates reached up to 1.7% PHB in dry weight. At least 70% of plant-produced hydroxybutyric acid was proven to be polymer with a molecular mass of up to 2,500 kDa. PHB synthesis levels of the transplastomic lines were decreasing when grown autotrophically but their phb transcription levels remained stable. Transcription of the three genes is divided into two transcripts with phbB being transcribed separately from phbC and phbA. In mature plants even low amounts of PHB were associated with male sterility. Fertility was only observed in a mutant carrying a defective phb operon. These results prove successful expression of the entire PHB pathway in plastids, concomitant, however, with growth deficiency and male sterility.
Keywords Nicotiana tabacum - Phb operon - Plastid transformation - Polyhydroxybutyric acid
Communicated by H. Lörz
Polyhydroxybutyrate (PHB) belongs to a class of polyesters of
3-hydroxy acids that are synthesized in various bacterial genera (Schubert et
Hai et al. 2001).
These polyhydroxyalkanoates (PHAs) are used as an energy source and also for
Materials and methods
The phb operon was cloned into transformation vectors by ligation of inserts, not by integration of amplified PCR products (compare Nakashita et al. 2001). Completeness and fidelity of the operon reading frame was verified by sequencing. This control of intactness was necessary in order to rule out damage to the operon during cloning. All cloning procedures were carried out using standard methods described in Sambrook et al. (1989). Nucleotide (nt) positions for transgene insertions as illustrated in Fig. 1 are given according to the plastome sequence data for EMBL accession no. Z00044 (Shinozaki et al. 1986).
Fig. 1. Map of the integrated phb operon in the plastome. The map shows the plastome insertion of the phb operon under control of the plastid psbA promoter (5-Plastid) and with original termination sequence from Ralstonia eutropha (3-Bact.). Location of the probe used for analysis (shown in Fig. 2) consisted of a 2.3 kb PstI-cut fragment (nt 1968–nt 4290 from start codon); restriction sites for ApaI are labelled A. Expected hybridization fragments for transformant lines are given at the bottom of the map. Intact transformants lines should contain a 4,743 bp and a 3,681 bp fragment
For integration of the phb operon with plastid expression
control elements, the transformation vector pKCZ was used (Zou 2001;
Huang et al. 2002).
This pUC19 based vector contains plastome homologous flanks including loci
trnN and trnR within the inverted repeat (IR) region. Plastome
insertion flanks for series P were located in IR-A (nt 109230–110348 and nt
110349–111520) and in IR-B (nt 131106–132277 and nt 132278–133396) respectively.
After PCR amplification by pfu polymerase the psbA promoter and
were linked to the 5 end of phbC in the pUC vector by ligation of
an NcoI-KpnI-cut fragment. This 5 region was
transferred to pHBR68 (Schubert et al. 1988),
the phb operon-containing vector by cut and ligation of a
BamHI-AgeI fragment. In a final step the operon with 5 psbA motifs
was excised by SmaI and XhoI and transferred to transformation
vector pKCZ cut by SacII and XhoI, after generating blunt ends
using Klenow fragment.
Nicotiana tabacum L. cv. Petit Havana plantlets
(Surrow Seeds, Sakskøbing, Denmark) were grown from seeds in vitro at 25°C
(0.5–1 W/m2 Osram L85 W/25 universal-white fluorescent
Analysis of transgenic lines by PCR and Southern hybridization
DNA was extracted from 100–150 mg in vitro or greenhouse plant material using the DNeasy plant DNA isolation kit (Qiagen, Hilden, Germany). PCR was carried out with the sense primer located in the transgene and the antisense primer positioned in the plastome outside the vector flanks. For Southern blots, 3 µg digested total plant DNA was separated on 0.8% agarose gels. Blots were prepared by transfer to nylon membranes (Hybond-N, Amersham). Specific probes were random prime labelled with 32P using Klenow fragment and hybridized to the membranes. Hybridization was carried out overnight at 65°C in Church buffer (0.5 M sodium phosphate, pH 7.5 and 7% SDS). Filters were also hybridized simultaneously with a probe derived from lambda DNA in order to detect size marker bands. Blots were washed twice at 50°C in 0.1% SDS and 2×SSC, pH7 for 30 min and once at 65°C in 0.1% SDS and 2×SSC for 30 min. Filters were exposed on imaging plates for 8 h and signals were detected using a phosphoimager (BAS-1500, Fuji, Tokyo).
Analysis of transgenic lines by Northern analysis
Total RNA was isolated from 30–100 mg plant material (leaves) using a RNeasy plant mini kit (Qiagen). About 5 µg RNA were separated on 1.2% formaldehyde-agarose gels and transferred to nylon membranes. Blotting was carried out as described by Sambrook et al. (1989). Random primed 32P-labelled DNA probes were hybridized to the membranes as described for Southern blotting. Membranes were washed with 0.1×SSC, 0.1% SDS at 65°C. Signal strength was determined using the phosphoimager.
PHB contents were measured in leaf material and callus. For monomer
determination, preextraction with ice-cold methanol was performed. PHB contents
were measured by gas chromatography using a 10m-CP-WAX-52CB column with a
diameter of 100 µm and 0.2 µm liquid phase. The procedure was adapted
to small volumes and a short GC column according to Brandl et al. (1988).
In order to produce PHB in plastids we transferred the phb operon from R. eutropha to the plastid genome of tobacco. For integration of the operon, a plastome locus within the inverted repeat regions was targeted. The map of the plastome insertion is shown in Fig. 1. In the experiments presented, the operon containing the promoter and 5-UTR of the plastid psbA gene was inserted between the plastid genes trnN and trnR. Five independent transformants P1-P5 were regenerated and analysed for their PHB contents.
After transformation, correct insertion and total length of the operon in the plastome of transformants was confirmed by PCR and Southern hybridizations. Probes and expected hybridization signals of phb cassettes are given in Fig. 1, and results are shown in Fig. 2. For intact insertions, 3.7 kb and 4.7 kb signals were expected, when hybridized with phbA and phbB probes. P1 to P5 exhibited this fragment pattern correctly. We also detected operon insertions being partially deleted; genotype P6 was a deletion mutant, which lacked 1.5 kb of the operon. Regenerates which showed deletions were excluded from further analysis.
Fig. 2. Proof of correct insertion by Southern hybridization. In order to show correct insertion of the phb operon, a specific PstI-BamHI fragment containing phbA and phbB genes was used as probe (nt 1968–nt 4334 from start codon). DNA was cut by ApaI. For wild type (WT) no signal is expected (nonspecific background band at 2.6 kb). Lanes 1–7 have DNA of series P1–P6. Lane 8 has wild type. Lane 9 has DNA standard. Sizes in kb are as follows: >20 kb, 9.8, 7.7, 6.2, 5.1, (4.3 faint), 3.5, 2.7, 2.4, 2.2, 1.9, 1.5, 1.3, 0.9. Intact transformants lines revealed a 4.7 kb and 3.7 kb signal. The sample of lane 6 shows a partial deletion of the integrated phb operon
As variable degrees of homoplasmy could affect PHB synthesis, it was necessary to analyse for residual wild type plastome. In order to test whether the phb operon becomes lost once spectinomycin selection pressure is removed, we transferred the transformants to the greenhouse to determine whether application of antibiotics was necessary for maintenance of the transgenic plastome. Following a 3-month greenhouse period, a further Southern analysis with the plastomic probe trnN revealed that plants were homoplastomic with respect to the operon insertion. Wild type plastome was no longer detectable when hybridized with plastid DNA flanking the operon insertion. Figure 3 shows the autoradiography of these findings.
Fig. 3. Southern hybridization for proof of homoplasmy. For proof that there is no heteroplasmy in plants with lower expression levels, we used as probe a plastome specific fragment containing trnR positioned at nt 109230–110348 within the direct repeat. Plant DNA was cut by ApaI. For wild type (Wt) a 2,649 bp signal is expected. Lane 1 has DNA standard. Lane 2 has wild type. Lanes 3–9 have DNA of series P1–P6. Lane 10 has DNA standard. For intact transformants, a 4.7 kb signal is expected. P6 is a transformant with partially deleted phb operon. The slope on the right side is due to a "smiling" effect of the Southern gel
Five confirmed transformant lines were analysed by GC, GPC and HPLC for their PHB contents.
PHB contents were measured by GC as shown in Fig. 4. In all determinations certain smaller peaks due to compounds with similar properties were found. In wild type tobacco we measured an average background level corresponding to 15 ppm in d.w. with a standard error of 3.6 ppm.
Fig. 4A–C. Gas chromatography of PHB contents. A Gas chromatographical analysis of in vitro leaf material from a young regenerate of transplastomic tobacco. B PHB standard. C Wild type tobacco. Intensity of peaks was measured in pA. The arrows indicate peaks corresponding to 3-hydroxybutyrate in the standard and in transplastomic line P1
Significant amounts of PHB in transformants
In vitro propagated PHB lines P1–P5 with intact phb reading frames under control of the psbA 5region were analysed for their PHB contents. The average of these transformants was 715 ppm in d.w. The different transformant lines revealed a high variability with regard to their PHB levels. Fig. 5 gives the contents in ppm in d.w. for the transformants and wild type tobacco at different developmental stages. Young leaves of the transformant lines P1 to P5 were analysed directly after regeneration from callus and a second time following a 3-week regeneration phase when plants could be transferred to the greenhouse. The transformants with clearly increased PHB contents revealed growth retardation in the early developmental stages. Lines that were derived from highly expressing genotypes showed severely decreased PHB contents after a period of further growth with increasing autotrophy.
Fig. 5. Average PHB contents at different developmental stages. Leaf material of each transformant line P1–P5 was analysed directly after regeneration from callus (A) and following a three weeks growth phase when plants were transferred to the greenhouse (B). PHB values were determined in dry matter from the transplastomic tobacco lines. Standard errors (SE) are given as vertical lines within the bars. Measurements for each genotype were repeated as follows: P1A 12, P1B 16, P2A 2, P2B 2, P3A 4, P3B 2, P4A 3, P4B 3, P5A 5, P5B 3
For example, young leaf tissue of line P1 accumulated up to 17,442 ppm in dry weight in single PHB determinations. This corresponds to 1.7% PHB. On further growth the average PHB content of transformant lines declined to 20 ppm.
For the most highly expressing plants, the main part of 3HB was proven to be polymer by dichloromethane extraction prior to gas chromatography. In line P1 with high PHB expression, the proportion of extractable high molecular weight PHB was at least 75% of the total PHB determined in dry weight. Pre-extraction with cold methanol precipitation gave an estimate of monomer content of less than 14%.
Fractionation within the extractable PHB by gel permeation chromatography and subsequent quantification by gas chromatography showed that the largest fraction existed as high molecular weight PHB in the range ca. 60 kDa to 3,000 kDa. Distribution of PHB polymer sizes is shown in Fig. 6. The maximum peak was located between 320 kDa and 840 kDa reflecting a polymerization of ca. 3,500–10,000 monomers.
Fig. 6. Polymer fractionation of PHB formed in a transplastomic plant. Gel permeation chromatography showed heterogeneity of polymer synthesis in transplastomic tobacco. Here, the PHB spectrum in line P1 is given. Dispersion was determined within extractable PHB. Polymer size is given separately as kDa. A size of 840 kDa reflects a polymerization of ca. 10,000 monomers
In order to trace back decreased contents within identical transformant genotypes, PHB determination in different tissues was carried out and a correlation between age of leaves and PHB accumulation in transplastomic plant tissue was observed. Figure 7 shows a gradient extending from lower contents in the youngest leaves to high PHB amounts in the basal leaves.
Fig. 7. Gradient of PHB contents within a transplastomic tobacco plant. PHB contents increased from upper leaves downwards. Values were measured in plants with the plastid operon. Standard errors are given as vertical lines
Northern analysis of PHB-expressing plants
To investigate the reasons for high variability in PHB synthesis, we analysed the transcription of the phb operon in the different transplastomic plants by comparing transcript expression using three probes as given in Fig. 8.
Fig. 8. Phb operon transcript arrangement deduced from Northern analysis. Probe phbAB consisted of a 2.3 kb PstI-cut fragment (nt 1968–nt 4290 from start codon). Probe phbB 3 with 0.6 kb (nt 3749–nt 4292 from start codon) was cut out by NcoI and EcoRI digest. Probe phbB 5 with 0.5 kb (nt 3111–nt 3669 from start codon) was amplified with PCR primers
Transcript analysis was performed within the plants containing the phb operon under control of the plastid promoter. DNA hybridization of greenhouse-grown transformants containing 20–50 ppm PHB with probe phbAB showed that the phb operon is expressed in two transcripts (Fig. 9): one transcript with a length of 3.2 kb containing phbC und phbA, and a second one with the length of 1.1 kb containing the phbB gene. Both the probe with the 3 phbB region and the 5 end of phbB visualized the same phbB transcript, as shown in Fig. 9C.
Fig. 9A–C. Comparison of RNA levels in PHB transformants. A Northern gel with lanes containing 10 µg total plant RNA each. Sizes of RNA standard are as follows: 6 kb, 4 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.2 kb. B Transcript signals detected with probe phbAB; hybridization shows a 3.2 kb transcript, containing phbC and phbA. C Transcript signals detected with probe phbB; hybridization shows a 1.1 kb transcript containing phbB
From the hybridization patterns detected we postulate the transcript arrangement given in Fig. 8. Variations in the RNA expression level between the genotypes of transformants were insignificant. There was no correlation to the variability found in their PHB synthesis. The genotype P1 with high PHB expression rates did not contain higher transcription levels than the transformants with low expression levels.
In additional Northern hybridizations, we found that in some cases the amounts of the two phb transcripts were present in a non-stoichiometric ratio. As shown in Fig. 10, plant RNAs extracted from two identical lines in the same developmental stage could exhibit different distributions in their transcript ratio between the 3.2 kb and the 1.1 kb fragment. However, there was no correlation to the PHB contents determined in these plants.
Fig. 10. Variable transcript distribution within RNAs of identical transformant lines. A non-stoichiometric ratio between the 3.2 and 1.1 kb transcript was detected in two different RNA extractions in lane 1 and 2 of P4 with probe phbAB
Developmental effects on transcript expression
In order to test if the early developmental stage of the plants has an influence on PHB expression, in a further experiment, we compared the early regeneration stage with the following growth stage, in which the PHB content was decreasing. The plants P1 to P5 compared in this Northern analysis differed in their age. In the first set RNA was extracted from these plants directly after regeneration from callus and in the second set RNA was isolated from 3-week-old in vitro plants.
This comparison revealed only small differences in the RNA expression of the respective lines as shown in Fig. 11. Transcript levels did not differ with the plants' developmental stage. No significant difference of RNA signals was detected by probing with phbAB.
Fig. 11. Comparison of RNA abundance in different developmental stages. The RNAs in lanes 1–5 were extracted from plants directly after regeneration from callus. RNAs in lane 8–12 were isolated from three weeks old in vitro plants prior to transfer into the greenhouse. The upper part of the figure shows the RNA gel used for blotting; the lower part shows the RNA signals after hybridization. Sizes of RNA standard are as follows: 6 kb, 4 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.2 kb. The Northern analysis was carried out with probe phbAB
Phenotype of PHB containing plants
Most of the regenerates with high PHB content grew slowly in comparison with wild type tobacco. We observed a strong negative correlation between PHB accumulation and plant growth. But even moderate levels of PHB could correlate with growth retardation as observed in line P3, for example, which contained 88 ppm PHB in the early regeneration phase. Eventually all genotypes grown in greenhouse appeared healthy. However all confirmed transformants were dropping at least 95% of their flowers and were male sterile in T0 and also in the T1 generation as shown in Fig. 12. By fertilization with wild type pollen we obtained seeds but out of the complete experiment, only genotype P6 with a partially deleted phb operon, which was serving as a control, bore completely fertile inflorescences.
Fig. 12A, B. Sterile and fertile PHB transformants. A Flowers of confirmed transformants as represented by P1 were completely sterile. These transformants developed seeds only after fertilization with wild type pollen. B Genotype P6 containing a partially deleted phb operon was fertile
Expression of the genes for bacterial PHB synthesis in plastids has
the advantage of decreasing the risk of unwanted gene escape to the environment
via pollen. In addition, site-specific integration of transgenes in the plastome
generally allows a more predictable transformation event compared to nuclear
transformation (Poirier et al. 1992;
Nawrath et al. 1994;
Houmiel et al. 1999;
Saruul et al. 2002;
Menzel et al. 2002).
Here we report on significant PHB synthesis mediated by plastome-localized
transgenes. Hydroxybutyric acid was produced in tobacco following transformation
with an expression construct under plastid regulatory elements. PHB synthesis in
transplastomic plants carrying these constructs was determined in five plants
and selected material showed a PHB content of up to 1.7% in d.w. At least two
thirds of the PHB was shown to be polymer with a molecular weight in a range
between 100 and 1,000 kDa. These PHB contents exceeded the values measured
in previously reported transformants by more than three orders of magnitude
(Nakashita et al. 2001).
The data given in this report are in the same range as those derived from our
wild type control. PHB content of transgenic lines was highly variable. In
addition, we found that subclones, which were initially characterized as highly
accumulating, could show severely decreased PHB contents. Surprisingly there was
a strong dependence of the variation of PHB synthesis on the developmental stage
and growth conditions. More PHB was observed in heterotrophically grown tissue
culture material than in plants transferred to the greenhouse. Transcript
abundance did not correlate with the variability in PHB synthesis. In addition
all positive lines showed growth retardation and male sterility.
Acknowledgements This work was supported through the Fachagentur für nachwachsende Rohstoffe (grant no. 99NR074). The excellent technical assistance by Katharina Yeiser, Stefan Kirchner and Petra Winterholler is gratefully acknowledged.