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Molecular Engineering of Phytoalexins in Plants: Benefits and Limitations for Food and Agriculture Philippe Jeandet,* Eric Courot, Christophe Clément, Sylvie Ricord, Jérôme Crouzet, Aziz Aziz, and Sylvain Cordelier Laboratory of Stress, Defense and Plant Reproduction, Research Unit EA 4707, SFR Condorcet FR CNRS 3417, Faculty of Sciences, University of Reims, P.O. Box 1039, 51687 Reims Cedex 2, France Increased disease resistance following foreign phytoalexin synthesis in a novel plant was first reported with the transfer of the STS grapevine gene into tobacco.3 Gene expression resulted in the production of resveratrol, whereas non transformed tobacco plants were found unable to synthesize it. From this pioneering experiment, a number of genetic transformations were then performed with the same STS grapevine genes or STS genes from other plant species using various host plants, including rice, tomato, alfalfa, kiwifruit, Arabidopsis, aspen, white poplar, barley, wheat, lettuce, soybean, hop, papaya, pea, apple, oilseed rape, strawberry, banana, and grape.4 Generally, phytoalexin-related gene overexpression resulted in increased resistance against pathogens in the genetically modified plants (Figure 1). Improvement of resistance was
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lants in their natural environment are facing large numbers of potentially pathogenic and beneficial microorganisms, mainly fungi and bacteria. To cope with these stresses, plants have evolved a variety of resistance mechanisms that can be constitutively expressed or induced. Phytoalexins, which are biocidal compounds of low molecular weight synthesized by and accumulated in plants as a response to stress, take part in this intricate defense system.1 Besides their role as defense compounds of plants, some phytoalexins have been shown to display health-promoting effects in humans, possessing cardioprotective, antitumor, antioxidant, antiaging, or neuroprotective properties. Therefore, the genetic manipulation of phytoalexin biosynthesis has been investigated for the purpose of increasing the resistance of plants to pathogenic microorganisms and improving food quality by enhancing antioxidant and/or anticancer activities in vegetables, cereals, or fruits. However, as some phytoalexins may be toxic for humans,2 one of the most pressing challenges is developing genetic strategies to reduce the negative impacts of phytoalexins on human health. Here we discuss the benefits and limitations of engineering phytoalexins in plants and offer new perspectives into human health promotion and plant disease control. The potential of phytoalexins to confer plants a resistance to pathogens has been evidenced in transgenic and mutant plants.1 Research has largely focused on resveratrol, the central phytoalexin for grapevines, the biosynthesis of which is catalyzed by stilbene synthase (STS). As this phytoalexin is produced in one step from precursor molecules present throughout the plant kingdom, the introduction of a single gene is therefore sufficient to produce resveratrol in heterologous plant species. © XXXX American Chemical Society
Figure 1. Stilbene synthase engineering in tomato: benefits for disease resistance to pathogens and for nutritional value.
observed in alfalfa to spring black stem, in rice to rice blast, in wheat and barley to gray mold and powdery mildew, in tomato to late blight, in wheat to leaf rust, in papaya to bud-rot, and in Arabidopsis to anthracnose.4 This highlights the potential of phytoalexin engineering as a promising alternative to the use of chemical pesticides. However, STS overexpression has also resulted in a lack of increased resistance to pathogens in some species. For example, no resistance was observed in transgenic poplar to rust disease or in kiwi plants to gray mold.4 Resveratrol accumulation in transgenic tobacco was found to delay but not abolish the infection process of leaves by gray mold. These examples point out limitations in the strategy of engineering phytoalexin synthesis in plants because the obtained resistance may be too weak or phytoalexins may be less efficient than chemical fungicides. The above inconsistent Received: March 1, 2017
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DOI: 10.1021/acs.jafc.7b00936 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
for humans should not compromise the ability of a plant to defend itself. Tissue-specific reductions of toxic phytoalexins in engineered plants will thus be needed.2 Additionally, tests will be required to detect unexpected consequences of metabolism alterations resulting from the genetic transformations.
results show that controlling pathogens in plants through exogenous expression of STS genes is feasible, but it remains largely empirical and not predictable, thus opening the way for further research. Molecular genetic engineering of phytoalexins in plants for disease resistance has been limited to exploiting only a few biosynthetic genes. Apart from STS gene transfer experiments, there have been some reports on genetic transformations with isoflavonoid phytoalexin genes. Manipulations with STS genes require only relatively simple genetic constructs with a single gene. In more complex biosynthetic phytoalexin pathways including numerous enzymatic steps, the main limitation is the impossibility of engineering the entire pathway. The challenge is to choose the appropriate enzyme catalyzing the limiting step or the entry point of the pathway. For example, genetic transformation of alfalfa with a gene encoding isoflavonoid-7-Omethyltransferase, a key enzyme in the biosynthesis of the phytoalexin maackiain, resulted in an increased resistance to spring black stem.5 As the genetic engineering of complex phytoalexin pathways is difficult, modulation of the regulatory factors of these pathways could instead represent original entry points for the regulation of phytoalexin biosynthesis.1,6 Phytoalexin biosynthesis is indeed up- or down-regulated by the synthesis of many endogenous molecules such as phytohormones, transcriptional regulators, defense-related genes, glycosyltransferases, or kinases. For example, genetic manipulation of isoflavonoid phytoalexin glycosylation (e.g., glycosylation of scopoletin in tobacco lines overexpressing an UDP-glucosyltransferase gene) was shown to be associated with a restriction of tobacco mosaic virus in leaves.7 In the same way, genetic manipulation of MAP kinases in transgenic Arabidopsis thaliana through a gain-of-function study resulted in 50 to 400-fold increases in the expression of the genes encoding P450 enzymes implicated in the formation of camalexin.6 Besides applying genetic transformations for disease resistance improvement, there have also been several studies on functional food application through the enhancement of the nutritional value of transgenic plants. However, research in this area has been limited to increasing the antioxidant and antiradical activities in STS-overexpressing tomato lines (Figure 1).4 Therefore, such results need to be extended to a number of other plants and phytoalexins. Engineering crops with phytoalexins can also pave the way for the valorization of underutilized agricultural byproducts. For example, a gene silencing strategy utilizing a targeted RNAi-approach dedicated to disrupting the toxic gossypol synthesis has been applied to cottonseeds. This study suggested that generating gossypol-free seeds could allow the use of cottonseed protein to feed half a billion people per year.2 Similarly, genetic manipulations of potato with an antisense invertase-coding gene significantly reduced the amounts of toxic phytoalexins such as the glycoalkaloids α-solanine and α-chaconine.8 To date, significant success in the engineering of phytoalexin biosynthesis has been obtained with resveratrol. Further research should address engineering with other phytoalexins. This may be achieved either by acting on the genetic modulation of the regulatory factors of the complex phytoalexin pathways or by introducing multiple transgenes for the coordinated expression of enzymatic activities. Future works should also focus on improving the nutritional quality of food products through genetic engineering of phytoalexins with interesting properties as well as down-regulating those potentially harmful to humans. However, reducing the level of a phytoalexin toxic
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AUTHOR INFORMATION
Corresponding Author
*(P.J.) Phone: +33-3-26913-341. Fax: +33-3-26913-340. E-mail:
[email protected]. ORCID
Philippe Jeandet: 0000-0003-3023-4871 Eric Courot: 0000-0001-9719-8604 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Jeandet, P.; Clément, C.; Courot, E.; Cordelier, S. Modulation of phytoalexin biosynthesis in engineered plants for disease resistance. Int. J. Mol. Sci. 2013, 14, 14136−14170. (2) Sunilkumar, G.; Campbell, L. M.; Pukhaber, L.; Stipanovic, R. D.; Rathore, K. S. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18054−18059. (3) Hain, R.; Reif, H. J.; Krause, E.; Langebartels, R.; Kindl, H.; Vornam, B.; Wiese, W.; Schmelzer, E.; Schreier, P.; Stöcker, R.; Stenzel, K. Disease resistance results from foreign phytoalexin expression in a novel plant. Nature 1993, 361, 153−156. (4) Delaunois, B.; Cordelier, S.; Conreux, A.; Clément, C.; Jeandet, P. Molecular engineering of resveratrol in plants. Plant Biotechnol. J. 2009, 7, 2−12. (5) He, X. Z.; Dixon, R. A. Genetic manipulation of isoflavone 7-Omethyltransferase enhances biosynthesis of 4′-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa. Plant Cell 2000, 12, 1689−1702. (6) Ren, D.; Liu, Y.; Yang, K. Y.; Han, L.; Mao, G.; Glazebrook, J.; Zhang, S. A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 5638−5643. (7) Gachon, C.; Baltz, R.; Saindrenan, P. Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance. Plant Mol. Biol. 2004, 54, 137−146. (8) Matthews, D.; Jones, H.; Gans, P.; Coates, S.; Smith, L. M. J. Toxic secondary metabolite production in genetically modified potatoes in response to stress. J. Agric. Food Chem. 2005, 53, 7766− 7776.
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DOI: 10.1021/acs.jafc.7b00936 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
