Many toxicants in tobacco smoke have their origins in the cellular structure of the tobacco plant, or might be affected by processes occurring in the plant during growing, senescence and curing, and so understanding the genes involved may help develop new plants that result in lower toxicants both in the tobacco and in cigarette smoke.
Our tobacco biotechnology research programme is carried out at our site in Cambridge. Here we are undertaking both basic research on the tobacco genome, and practical plant modification research which includes some field trials of GM plants in the United States.
In order to give us guidance on which genes may be involved in the formation of toxicants in tobacco and smoke we have, in collaboration with the French tobacco company Altadis, generated new sequence data as a part of the European Sequencing of Tobacco Project (ESTobacco). We have combined these data with other published data to develop a tobacco gene chip.
This Tobacco Microarray, produced in conjunction with the company Affymetrix, contains probes for around 43,723 tobacco sequences and allows us to investigate the expression of all these genes.. Our initial research has found that we are able to measure differences in gene expression between (i) tissue at different stages of growth, (ii) different tissues or (iii) tissue exposed to different treatments. This will enable us to better understand gene expression changes in relation to fundamental processes within tobacco.
For example, in a study of gene expression during senescence we have used microarray analysis to reveal gene expression changes during dark induced senescence in leaves from Nicotiana tabacum. Senesence is the last phase of plant development and is a highly ordered, dynamic and regulated process, where chlorophyll breaks down and tobacco leaves change from green to yellow – it is one of the plant processes that may affect precursors of tobacco toxicants. Our initial study found that eleven genes had a greater than two-fold up-regulation of expression during mid to late senescence.
We are also working on the development of a Tobacco Expression Atlas (TobEA), a database of gene expression in different tobacco organs at different plant developmental stages.
The below figures show a normalised gene expression map in initial eight TobEA organs (Figure 1) and a Venn diagram of genes expressed in shoot, root and imbibed seed samples (Figure 2).
We hope that this effort will provide a useful reference for academic researchers investigating tobacco modification and aid plant functional genomics and systems biology research.
In order to change the metabolites present in a tobacco leaf, the plant is genetically modified. This approach uses techniques such as cloning and transformation to add or remove a specific gene and differs from traditional breeding where genes from two individual parents are randomly mixed
Once a target gene for modification has been identified, it is isolated, cloned into an appropriate vector containing a suitable promoter and antibiotic resistance and transformed into Agrobacterium. This natural plant pathogen facilitates genetic modification by integrating its own DNA into the plants. Leaf discs are cut from a tobacco plant and incubated with the Agrobacterium before plating onto media to promote the development of a callus.
A callus is a mass of undifferentiated cells that can be made to differentiate into the specialised tissues of a whole plant by the addition of appropriate hormones. Not all plant cells will be transformed with the target gene so the medium also contains an antibiotic that only allows the growth of transformed cells. The genetically transformed plants can often contain multiple copies of the target gene, which can adversely affect its ability to function so all new transgenic plants are analysed for copy number by Real-Time PCR in order to produce genetically stable homozygous lines.
Transgenic populations are then assessed for the appropriate biochemical changes. This can include qualitative or quantitative enzyme or metabolite assays. Transgenic plants that show the expected biochemical changes are then examined further under field trial conditions.
For example, in one study we have sought to increase the nitrite reductase (NiR) activity in order to decrease the residual nitrite and nitrate. Nitrate and nitrite levels in leaf may affect the levels of tobacco-specific nitrosamines found subsequently in smoke. In this work we used the A. thaliana NiR gene placed under the control of a constitutive promoter, carnation etched ring virus to transform tobacco plants. The transformed plants had a phenotype exhibiting a persistence of leaf greenness, which was particularly apparent as the population matured.
In the upper sink leaves of the T1 homozygous AtNiR population, NiR activity was increased by up to four-fold and NR activity was increased six-fold, implying that increased NiR activity also affected NR activity. Nitrite content appeared to be reduced in the transgenic lines but the nitrate content did not show any distinct difference. Even so, the phenotype observed was the same as that seen in the primary transformed population, where mature leaves demonstrated a stay-green phenotype.
The behaviour of the plant can be quite different when grown in the field as compared to when grown in a greenhouse. So an important aspect of our research is to undertake field trials. They enable us to assess the performance of our new tobacco varieties when grown under commercial field conditions and provide large quantities of cured leaf material for extensive analysis. We can produce on average 7.5kg of cured leaf per new variety for analysis and comparison with commercial and internal controls.
Our current field trials are carried out in North Carolina, USA, in association with local farmers who have contracts with the tobacco company RJ Reynolds. The farmers both grow and cure the tobacco. One reason to choose the US for field trials is the fact that the US has clear regulations in place for GM trials. Every trial we undertake is approved by APHIS and USDA and open to inspection. The tobacco plant typically is topped, reducing the potential for any cross-pollination, but as a further precaution our trials are also run with plants that are male sterile.