The first step in plant transformation consists in the entrance of a desired genomic sequence into a plant cell. Stable nuclear transformation is caused by integration of the recombinant DNA in the nuclear genome. DNA can be transferred into the nuclear genome by either direct (e.g. biolistics) or indirect (e.g. Agrobacterium) methods, it depends on the plant species and the type of tissue (Thanavala et al., 2006).
In the stable nuclear transformation whole plants can be regenerated, eventually producing a seed stock or a plant tissue maintained in an aseptic culture. The advantage of this system is that the transgene is heritable, permitting the establishment of a seed stock for future use. Establishment and characterization of stable transgenic lines can be costly and time consuming. Large numbers of transgenic lines need to be screened and analyzed before a single optimal line can be selected for protein production (Ling et al., 2010). Other disadvantages are gene silencing and position effects.
Nuclear transformation has been employed and extensively studied in many plant species, however, it generally results in low expression of soluble foreign proteins (Yap & Smith, 2010).
Recombinant proteins can be targeted to different subcellular compartments in plant cells, such as cytostol, apoplast, endoplasmic reticulum, vacuole or chloroplast.
2.3. Optimization of expression level
Increasing the transcription rate of stably transformed gene sequences is the most direct and efficient approach to increase protein expression. This is mainly achieved with the use of a strong constitutive or inducible promoter. Constitutive promoters directly drive the expression in all plant tissues and are independent of the production host developmental stage. The best known and most widely used constitutive promoter in plant biotechnology is derived from Cauliflower Mosaic Virus (CAMV35S). It is more effective in dicots than monocots. Alternative constitutive promoters frequently used in plant cell transformation are the ubiquitin promoter, histone H2B promoter and the (ocs)3mas promoter (Hellwig et al., 2004). The ubiquitin promoter, isolated from a variety of plants including maize, Arabidopsis, potato, sunflower, tobacco and rice, has been frequently used to express biopharmaceuticals in plant cells. The (ocs)3mas promoter, constructed from octopine synthase (osc) and mannopine synthetase (mas) agrobacterial promoter sequences, was used for the expression of Hepatitis B antigen in a soybean cell culture (Smith et al., 2002). Other constitutive promoters used for expression of foreign genes in transgenic plants include: tobacco cryptic constitutive promoter (Menassa et al., 2004), Mac promoter which is a hybrid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer region (Dai et al., 2000), rice actin promoter (Huang et al., 2006), banana actin promoter (Herman et al., 2001), C1 promoter of cotton leaf curl Multan virus (Xie et al., 2003), nopaline synthase promoter (Stefanov et al., 1991).
Inducible promoters allow external regulation by chemical stimuli such as alcohol, steroids, salts, sucrose or environmental factors such as temperature, light, oxidative stress and wounding. Inducible expression is advantageous as this allows protein production to be separated from cell growth. The use of chemical inducible promoters in combination with the chemical responsive transcription factor can further restrict the target transgene expression to specific organs, tissues or even cell types (Zuo & Chua, 2000). The examples of inducible promoters and synthetic transcription activators are: the rice α-amylase 3D (RAmy3D) promoter, which is induced by sucrose starvation; the oxidative stress-inducible a peroxidase (SWAPA2); an estradiol-inducible chimeric XVE transcription activator and dexamethasone-inducible pOp/4v transcription activator (Xu et al., 2011), hydroxyl-3-methylglutaryl CoA reductase 2 promoter, which is inducible by mechanical stress (Cramer et al., 1996).
Tissue-specific promoters control gene expression in a tissue or in a developmental stage specific way. The transgen driven by such a promoter is expressed in a specific tissue leaving all the other tissues unaffected. It helps to force transgene expression in storage organs like seeds, tubers or fruits. Several of such promoters were tested: tuber specific patatin promoter (Jefferson et al., 1990), fruit specific E8 promoter (Jiang et al., 2007), arcelin promoter (Osborn et al., 1988), maize globulin 1 promoter (Rusell & Fromm, 1997), 7s globulin promoter (Fogher, 2000), rice glutelin promoter (Wu et al., 1988) and soybean P-conglycinin subunit promoter (Chen et al., 1986).
The optimization of promoters activity can be further improved by means of engineered DNA elements - enhancers, activators or repressors located up or downstream of the core promoter. Enhancers are shown to increase gene expression when placed proximally to the promoter, they bind activator proteins and promote RNA polymerase II placement at the TATA box. Transcription is also enhanced with flanking the transgene by nuclear scaffold/matrix attachment regions (S/MARs) important for structural organization of eukaryotic chromatin (Halweg et al., 2005).
The translational efficiency of a transgene is determined by proper processing (capping, splicing, polyadenylation, nuclear export) and mRNA stability. The 5’ and 3’ untranslated region (UTR) of the plant mRNA plays crucial roles in its processing (Cowen et al., 2007). The 5’-UTR is very important for 5’ capping and enables translation initiation, the 3’-UTR is indispensable in transcript polyadenylation which in turn influences the stability of mRNA (Chan and Yu, 1998). These untranslated sequences can be manipulated for the optimization of protein expression.
As the protein is synthesized, it undergoes several modifications before final delivery to its cellular destination. These modifications include enzyme involving glycosylation, phosphorylation, methylation, ADP-ribosylation, oxidation, acylation, proteolytic cleavage and non-enzymatic modifications like deamidation, glycation, racemization and spontaneous changes in protein conformation (Gomord & Faye, 2004). Post-translational proteolysis can be effectively minimized by targeting the foreign proteins to sub-cellular compartments such as the endoplasmic reticulum (ER). Proteolysis is more likely to occur in the apoplast and cytosol. ER retrieval signal (e.g. KDEL, HDEL) retains the expressed protein in the ER lumen and has been used to improve foreign protein stability. The ER contains many molecular chaperones facilitating nascent proteins folding or assembly and it is regarded as an ideal compartment for accumulating many classes of foreign proteins (Nuttal et al., 2002).
Other strategies for proteolytic degradation reduction are: co-expression of recombinant protein and protease inhibitors, co-expression of protein co-factors or subunits, knockout mutations in the genes encoding specific proteolytic enzymes.
The recent advent of highly efficient transient expression systems has completely changed the concept and revolutionized plant made pharmaceutical research. Transient transformation implies the expression of foreign DNA which cannot be inherited but is still transcribed within the host cell in a transient manner. Transient gene expression provides a rapid alternative to the time consuming stable transformation methods. This approach uses the plant hosts - Arabidopsis thaliana, Nicotiana tabacum, Nicotiana benthamina, Lactuca sativa. Transient expression of recombinant proteins in plants is performed by the use of engineered plant viruses and/or Agrobacterium mediated DNA transfer (agroinfection/agroinfiltration). Fast and high level expression is the major advantage of the transient expression systems. Full expression of a gene of interest in agroinjected leaves may be achieved in 3-4 days after infiltration with Agrobacteria. This system is simple and experimental procedures do not require expensive supplies and equipment. Leaves of greenhouse grown plants are infiltrated using a syringe without a needle, vacuum infiltration or the wound and agrospray inoculation method (Medrano et al., 2009). Supplementation of the infiltration media with Silwet L-77, Tween-20, or Triton X-100 improves the efficiency of transformation. In the transient expression system one can use different virus types: Tobamoviruses, Potexviruses, Potyviruses, Bromoviruses, Comoviruses and Gemniviruses. Prolific production of any given protein using the plant virus approach results from the fact that a virus can infect a plant systemically by moving in its symplast. The Agrobacterium based method involves the injection or vacuum infiltration of whole plants or their parts with a suspension of bacteria harboring the construct of interest (Gómez et al., 2009). Agrobacterium delivered plant viral vectors use the RNA polymerase II mediated nuclear export route including 5’ end capping, splicing and 3’ end formation. Plant RNA viruses replicate in the cytoplasm and are not adapted to nuclear splicing machinery which recognizes and removes cryptic introns from viral RNA leading to its degradation. The Agrobacterium delivered so called ‘first generation’ TMV and PVX vectors have low production capacity and require coinjection of a plasmid encoding gene silencing suppressor such as tombusvirus p19 or potyvirus P1/HC-Pro (Komarova et al., 2010).
A major breakthrough in viral expression strategies was facilitated by the recent advent of deconstructed virus vectors. Originally reported for the TMV-based magnICON system developed by ICON Genetics GmbH merges advantages of Agrobacterium-mediated DNA delivery and upgraded TMV based vectors where putative cryptic splice sites were removed and multiple plant introns inserted. Thus the basic idea is to amplify the foreign gene delivered by Agrobacterium tumefaciens to multiple areas of the plant allowing the virus to replicate and spread. In this process, bacteria start initial infection delivering the T-DNA encoded viral replicon to the nuclei of a large number of cells. Then, the transcripts are transported to the cytoplasm where the viral RNA amplification renders high yields of the desired protein (Gleba et al., 2005).
In conclusion, the two major strategies for expressing proteins in whole plants are transient expression with viral vectors and stable transformation where transgenes are targeted to either the nuclear or chloroplast genome. Stable transformation offers the advantage that protein production is scalable to large field production methods. However, this can be offset by low expression levels and the long time required for creating expressor lines stable across multiple generations. Today’s most promising direction in the referred field is emerging from synthesis of genetically engineered agrobacteria, viruses and plants in one precisely tailored system where synthetic and system biology meet each other.