Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production
- James R Petrie†1,
- Pushkar Shrestha†1,
- Qing Liu1,
- Maged P Mansour1,
- Craig C Wood2,
- Xue-Rong Zhou2,
- Peter D Nichols1,
- Allan G Green2 and
- Surinder P Singh1Email author
© Petrie et al; licensee BioMed Central Ltd. 2010
Received: 25 January 2010
Accepted: 11 March 2010
Published: 11 March 2010
Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed.
In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV) 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis.
The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.
The P19 silencing-suppressed N. benthamiana assay has previously been used to transiently-express an entire functional DHA biosynthesis pathway in leaf tissue by mixing and co-infiltrating Agrobacterium strains harbouring single-gene 35S-driven constructs . Although successful in identifying gene combinations capable of directing DHA synthesis, this approach could not be used to validate corresponding constructs destined for use in oilseeds since the genes in these constructs would be driven by seed-specific promoters that are not active in N. benthamiana leaf tissue. It is worth noting that seed-specific promoters are often used in a seed-trait engineering context both to obtain good expression and to limit the modified metabolites to the seed. This is especially important when engineering unusual oil pathways since the presence of these fatty acids in vegetative tissue can be deleterious to membrane structure and function.
Precise metabolic engineering of complex pathways requires the optimisation of multiple steps - a challenging prospect when limited to stable transformation and analysis of seed-expressed traits. We were therefore interested in modifying the N. benthamiana assay to allow the activation of seed-specific promoters in the leaf tissue. We hypothesised that extending the N. benthamiana leaf assay with the A. thaliana LEC2 transcription factor might allow rapid functional assessment of a complex construct designed for seed-specific expression in a land plant. The LEC2 transcription factor is currently the subject of considerable research due to its high-level control of entire metabolic pathways and LEC2 expression is known to establish a cellular environment that promotes the broad metabolic changes involved in embryo development [6, 7].
In this study we demonstrate the use of a LEC2-extended N. benthamiana transient assay in which the leaf expression of five genes driven by seed-specific promoters contained within a single construct resulted in the synthesis of the omega-3 polyunsaturated fatty acid DHA. The ability to rapidly express complex seed-specific constructs in leaf tissue will be increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways.
Results and Discussion
Identification of an efficient DHA synthesis pathway
Nicotiana benthamiana leaf fatty acid profiles
35S DHA mix
15.9 ± 0.2
16.6 ± 0.1
13.3 ± 0.1
13.2 ± 0.6
1.7 ± 0.1
1.5 ± 0.1
1.3 ± 0.1
1.1 ± 0
6.3 ± 0.3
5.6 ± 0.1
7.1 ± 0.3
7.5 ± 0.4
3.6 ± 0.3
3.3 ± 0.1
1.8 ± 0.1
2.4 ± 0.3
2.8 ± 0.1
2.8 ± 0.2
1.1 ± 0.1
1.5 ± 0.2
18.7 ± 0.1
13.0 ± 0.1
13.8 ± 0.1
12.7 ± 0.4
45.6 ± 1.4
40.2 ± 0.5
56.3 ± 0.7
44.8 ± 2.1
1.3 ± 0.4
0.6 ± 0
0.3 ± 0
0.5 ± 0.1
New ω6 PUFA
2.1 ± 0.2
2.4 ± 0.1
0.2 ± 0.1
0.2 ± 0.1
0.2 ± 0
New ω3 PUFA
2.0 ± 0
0.9 ± 0.1
1.2 ± 0.1 (15% Δ6-des)
0.4 ± 0
2.0 ± 0.1
0.3 ± 0
0.6 ± 0
2.3 ± 0
1.7 ± 0.1
2.5 ± 0.1
2.5 ± 0.2
Total new FA
All genes were found to be active and DHA was produced by the transgenic pathway. Use of the highly efficient P. cordata Δ5-elongase resulted in very low EPA accumulation in total leaf lipids (0.3%) with 94% being elongated to DPA. Importantly, this pathway resulted in the synthesis of very low levels of intermediate ω3 fatty acids due to the high conversion efficiencies achieved by the enzymes. There was also an almost complete absence of ω6 fatty acids due to the ω3-preference displayed by the M. pusilla Δ6-desaturase. This N. benthamiana leaf assay was useful in identifying a combination of genes that resulted in efficient DHA production but was seriously limited by requiring the use of independent, 35S-driven, genes. A pathway in this configuration is not useful in a stable seed context so we next attempted to extend the leaf assay to allow the analysis of such complex, seed-specific, constructs.
LEC2-enabled transient-expression of seed-specific promoters in leaf
Rapid validation of a complex seed-specific construct in leaf
The five genes comprising the efficient DHA synthesis pathway identified above were built into the complex seed-specific construct pJP3057 (Fig. 2D) which was then validated in the LEC2-extended N. benthamiana leaf assay. Production of DHA was observed when pJP3057 was co-infiltrated with 35S:LEC2 and 35S:P19 whereas the infiltration of 35S:P19 and either pJP3057 or 35S:LEC2 alone did not result in any LC-PUFA production (Table 1). Whilst these results were no guarantee of the success of the construct in a stably-transformed event, they did indicate that there was nothing fundamentally unstable in the structure of the construct.
Stable Arabidopsis transformation
Transgenic Arabidopsis seed fatty acid profiles
New ω6 PUFA
New ω3 PUFA
0.4 (18% Δ6-des)
0.6 (90% Δ6-elo)
0.2 (83% Δ5-des)
0.3 (93% Δ5-elo)
2.4 (89% Δ4-des)
Total new FA
We have demonstrated that it is possible to transiently-express genes driven by seed-specific promoters in leaf tissue to yield a functional metabolic pathway. The profound seed-like metabolic changes caused by the LEC2 transcription factor make the LEC2-extended system a more relevant background in which to assay genes and constructs destined for stable seed transformation than a traditional N. benthamiana leaf assay . It is worth noting that further work is required to define the extent to which this assay could be useful in predicting construct function in a stably-transformed context. It will also be interesting to determine which seed-specific promoters can be similarly activated by LEC2 in leaf tissue. We expect this new assay will have multiple applications, not least of which will be to provide a 'rapid-fail' test for poorly designed seed-specific constructs that would traditionally take at least one plant generation to assess. As genetic engineering moves from the manipulation of single genes to engineering of complex pathways, an assay system such as described here will be invaluable in accelerating the rate of optimisation of engineered pathways.
Binary vector construction
Each of the individual 35S constructs was built by inserting an Eco RI-flanked gene coding region into the same site of a pORE04 binary vector with already contained the A. tumefaciens NOS polyadenylation signal and modified by the addition of a double CaMV-35S promoter at the Sfo I site . The binary vector pJP3057 was built by first cloning the same Eco RI-flanked gene coding regions into the same site in an intermediate cloning vector between a truncated Brassica napus napin promoter, FP1, and the A. tumefaciens NOS polyadenylation signal. The entire cassette was then cloned into suitably adapted sites of the multiple cloning site in pORE04. 35S:P19 and the intron-interrupted, secreted GFP gene used in FP1:GFP was provided by Dr Peter Waterhouse.
N. benthamiana leaf infiltration
Each Agrobacterium tumefaciens strain AGL1 harbouring a binary vector was grown at 28°C with shaking in LB broth supplemented with the appropriate antibiotics for two days. The amount of culture required to yield 1 mL of OD600 nm = 2.5 culture was centrifuged (10,000 g, 1 minute). After removal of the supernatant the pellet was gently resuspended in 1 mL of infiltration buffer (5 mM MES, 5 mM MgSO4, pH 5.7, 100 μM acetosyringone freshly added) and the culture incubated at 28°C with shaking for a further three hours. Each culture was then used as a 10× stock for culture mixture, with the remainder of the required volume made up by infiltration buffer. All N. benthamiana infiltrations included a 35S:P19 culture. The culture mixtures were infiltrated as described by Voinnet et al. , into the underside of leaves of approximately one month old N. benthamiana plants that had been housed in a 23°C plant growth room with 10:14 light:dark cycle but moved to 28°C with water two hours prior to infiltration. Following infiltration the infiltrated regions were circled with a permanent marker and the plants were left at 28°C for one hour after which they were transferred to a 24°C plant growth room for five days before being harvested using a leaf disc cutter.
A. thaliana transformation
A. thaliana (ecotype Columbia) was used for plant transformations. Agrobacterium-mediated transformation was performed by the floral dipping method . T1 seeds were harvested and plated on media containing 20 mg L-1 kanamycin to test segregation ratios.
Total lipid extraction, lipid class analysis, fatty acid methyl ester preparation and all analyses were performed as previously described .
We thank Danny Holdsworth, Anne Mackenzie, Lijun Tian and Adam White for their excellent technical assistance.
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