The study aims to gain a better understanding of the effects of strigolactones on primary and secondary metabolism of medicinal plants. With the exception of their effect on lateral branching control, the influence of strigolactones in plants remains poorly understood. Arabidopsis thaliana will be used as a model system to which primary and secondary metabolite effluxes can be compared.
Transgenic hairy-root cultures of Sutherlandia frutescens and Arabidopsis thaliana will be used to study the synthesis of the secondary metabolites. A complex mixture of secondary metabolites accumulate in these plants and the effect of growth promoting factors on primary and secondary metabolite production will also be studied. The influence of strigolactones and/or auxin on both primary and secondary metabolism will be monitored in S. frutescens and A. thaliana hairy root cultures. Where possible, pharmacological studies will be performed to identify the active chemical compounds in Sutherlandia and Arabidopsis hairy root cultures that may be upregulated due to strigolactone and/or auxin application.
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Growth studies of Arabidopsis thaliana, Sutherlandia frutescens and Salvia stenophylla species will also be investigated with supplemented strigolactones and/or auxin. The effect strigolactones and/or auxin have on branching and root production of these plant species will be inspected as well as the physiological performance of these plants will be measured. This will enable us to perceive the effect which strigolactones and/or auxin have on growth and development and photosynthesis.
After this study the combined effect of strigolactones and auxin on growth and development as well as on plant metabolism of transgenic hairy root cultures and in vitro cultures will be apparent.
Model plant species have established an useful source to investigate the effect of various factors on the growth and development of plants and these effects on gene expression. Non-model plant species can be correlated to model plant species to better understand their reactions to certain stimuli. South Africa's rich diversity of plant species, especially medicinal plant species, have attracted much scientific and commercial attention to treat newly developed and well-known diseases or conditions like HIV/AIDS, cancers, stress and diabetes. Medicinal plants have been used for centuries by traditional healers and recently this has attracted the attention of researchers to study the compounds that are responsible for these healing abilities.
Sutherlandia (Lessertia) frutescens
Sutherlandia frutescens (also known as the cancer bush) has been used in traditional medicine in different cultural groups in southern Africa for centuries, dating back to the mid 1800's. Recently, many claims have been made on the efficacy of Sutherlandia extracts in helping to heal diseases such as internal cancers, HIV and AIDS, diabetes, stress and anxiety, inflammation, pain and wounds (Van Wyk and Albrecht, 2008) and a company called Phyto Nova (Pty) Ltd. initiated large-scale cultivation of Sutherlandia and the production of tablets and gels (Phyto Nova website, www.phyto-nova.co.za).
Canavanine, which is present in Sutherlandia, is a promising anti-cancer agent, particularly for treatment of pancreatic cancer (Crooks and Rosenthal, 1994). Ortega (2003) presented the role of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in inhibiting the migration of in vitro tumor cells. It has also been suggested that GABA may play a role in the improvement of the mood and well being, and this can indirectly reduce wasting in HIV/AIDS patients (Tai et al., 2004).
Bates et al. (2000) tested the effect of D-pinitol on the blood sucrose homeostasis of diabetic mice and concluded that pinitol exerts an acute insulin-like antihyperglycaemic effect but does not augment the effect of insulin.
Sutherlandia extracts have been shown to have anti-HIV characteristics, but the compound(s) that fulfil this effect is still unknown (Harnett et al., 2004). Harnett et al. also concluded in their study that the compounds that have the anti-HIV effects are not tannins or sulphated polysaccharides, as has previously been suggested.
The anti-inflammatory property of Sutherlandia extracts can be credited to the antioxidant ability of the extracts. A study by Fernandes et al. (2004) demonstrated that the antioxidants have superoxide and hydrogen peroxide scavenging abilities, which may contribute to the anti-inflammatory effect of hot water extracts of Sutherlandia frutescens subsp. microphylla powdered plant material.
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Growth promoting substances
Strigolactones are a group of terpenoid lactones thought to be derived from carotenoids, which inhibit shoot branching of plant species (Klee, 2008). Strigolactones also stimulate seed germination of parasitic plants and stimulate symbiotic fungi for mycorrhizal interactions (Gomez-Roldan et al., 2008). The strigolactones identified thus far have a similar four-ring backbone and differ from one another on the basis of the saturation of the rings (Gomez-Roldan et al., 2008; Umehara et al., 2008). The tricyclic lactone (A, B and C rings) are connected to a α,β-unsaturated furanone moiety (D-ring) via a enol-ether bridge (Humphrey et al., 2006). Zwanenburg et al. (2009) believed that the enol-ether bridge and the furanone ring (D-ring) are vital for the activity of strigolactones. The furanone ring stimulates the nucleophilic attack by an electron rich species. The D-ring is eliminated and the ABC part binds covalently to the receptor. Nijmegen-1 (Figure 1) on the other hand only has three rings (open C-ring), but the same α,β-unsaturated furanone ring and the enol-ether bridge. Thus the furanone ring can stimulate the nucleophilic attack.
Figure 1: Chemical structure of GR24 and Nijmegen-1 which are important for the mode of function of strigolactones. (Zwanenburg et al., 2009)
In our laboratory it has been found that GR24 increases the biomass and growth of Nicotiana benthamiana seedlings and in this study it will be tested if GR24 has a similar effect on the biomass and growth of hairy root cultures. They also found that GR24 treatment resulted in an increase of sucrose which is a primary metabolite and it is thought that strigolactone analogues may have an effect on other primary metabolites and perhaps even secondary metabolites. The synthetic strigolactone, GR24, also causes an increase in the ability to respond to stress. The possibility thus exists that there is an increase in the production of secondary metabolites, because secondary metabolism gets up-regulated under stress conditions.
Umehara et al. (2008) found that strigolactones or other downstream metabolites inhibit branching in rice, but they do not know how these strigolactones are further metabolized in plants. Gomez-Roldan et al. (2008) concluded in their study that the novel hormone which is responsible for inhibiting shoot branching belongs to the strigolactone family.
Plant Natural Products
Plants produce primary and secondary metabolites. Primary metabolites are essential for the survival of the plant, whereas secondary metabolites are produced for their antibiotic, anti-fungal, anti-viral and UV protection effect (Bourgaud et al., 2001). Secondary metabolites are of large interest because they serve as an important source of pharmaceuticals. Secondary metabolites are usually classified according to their biosynthetic pathways for example phenolics, terpenes and steroids, and alkaloids (Bourgaud et al., 2001).
Some of the key metabolites that accumulate in Sutherlandia frutescens include asparagine, proline, canavanine, γ-aminobutyric acid (GABA) and pinitol, with flavonol glycosides and triterpene glycosides (Van Wyk and Albrecht, 2008).
2. Aims and Objectives
The aim of this study is to successfully evaluate the mechanisms that control the synthesis of Sutherlandia's natural products and the role of strigolactones in both primary growth and secondary metabolite differentiation. These products will be evaluated in vitro by means of Agrobacterium rhizogenes-mediated transformation of S. frutescens cultures to up-regulate secondary metabolite production. A variety of analytical tools and bioassays will be used to study the chemical compounds and the secondary metabolites' mode of action.
The objectives of this study include:
Exposure of plant material to the growth promoting substances, strigolactones and/or auxin
Analysis of primary and secondary metabolites of model and non-model plant species
3. Materials and Methods
3.1 Plant material
3.1.1 Plantlet Culture Conditions
Nodal explants with axillary buds of Sutherlandia frutescens (2 cm) will be cultured on plant growth regulator (PGR)-free Murashige and Skoog (MS) media (Murashige and Skoog, 1962). The MS media is made up of 30 g/l sucrose, and 0.1 g/l myo-inositol and pH is adjusted to pH 5.7. The cultured explants will then be incubated for 16 hours in the light at a light intensity (PPFD) of 50 µmol.m-2.s-1 and then in the dark for 8 hours. These conditions will be maintained for a 30 day period, whereafter explants will be subcultured onto fresh media in a 15 cm glass culture vessel.
3.1.2 Hairy Root Cultures
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The hairy root cultures of Sutherlandia will be cultured on both on solid and liquid suspension PGR-free MS media (pH 5.7). Hairy root cultures are cultured in the dark for optimal root formation.
3.2 Effect of Strigolactone analogues
The effect of two synthetic strigolactone analogues, GR24 and Nijmegen-1, on the production of the complex compounds of Sutherlandia frutescens will be investigated via TLC, HPLC, LCMS and GCMS. The strigolactone analogues, at a concentration of 10-7 M, will be tested on 15 nodal explants, 15 hairy root solid cultures and 15 hairy root suspension cultures (15 control plants of each of these plant cultures will be used). The effect of the growth promoting factors on growth will be measured by determining the fresh and dry masses of the hairy root cultures.
3.3 Chromatographic analysis
Thin layer chromatography (TLC) can be used to identify the different compounds that are synthesized by the different plant cultures. The analytes, suspended in a mixture of toluene:diethyl ether:1.75 M acetic acid (1:1:1), will be separated on a silica gel (SA Health Info website, http://www.sahealthinfo.org/traditionalmeds/monographs/sutherlandia.htm). The TLC analysis can be followed by two-dimensional TLC to analyse secondary metabolites (Ciesla and Waksmundzka-Hajnos, 2009).
High pressure liquid chromatography (HPLC) can be used as a method for metabolite profiling of plant extracts (Grata et al., 2009). Liquid chromatography-mass spectrometry (LCMS) is a method which is used to identify and quantify primary metabolites such as flavonoids (Hughey et al., 2008), whilst gas chromatography mass spectrometry (GCMS) can be used for the identification and quantification of volatile secondary metabolites (Santos and Galceran, 2003).
3.6 Statistical analysis
For statistical analysis of the effect of the GR24 and Nijmegen-1 strigolactone analogues on the hairy root and nodal explant cultures ANOVA test will be used. ANOVA tests are used to test if multiple samples are alike (Akritas and Brunner, 2003). Local and global parametric tests will be done to look at the difference of each test individually and all the tests simultaneously (Tricot, 1989).
4. Possible Outcomes
The secondary metabolites mode of action will be investigated for a more clear understanding for research and industrial purposes. Exogenous application of strigolactones may also result in an increase in the levels of various valuable primary and secondary metabolites.