A metabolomic approach to thrips resistance in tomato
Romero González, R.R.
Citation
Romero González, R. R. (2011, October 11). A metabolomic approach to thrips resistance in tomato. Retrieved from https://hdl.handle.net/1887/17920
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Summary and general discussion
Western flower thrips (WFT), Frankliniella occidentalis, is one of the most serious crop pests worldwide, whose control represents a major challenge for both horticulture and floriculture. The development of insecticide resistance and the unsatisfactory performance of existing control methods puts a lot of pressure on pest management for the introduction of new and complementary crop protection strate- gies against WFT, among which host plant resistance remains as the ideal alternative. In this context the goal of the project was to scrutinize the genus Solanum searching for the bases of thrips resistance in tomato.
The workings of different potential resistance mechanisms against WFT in tomato were iden- tified, contrasted and evaluated in this thesis. In general the bases of thrips resistance in tomato were unveiled and described. The objective of this project was therefore successfully and satisfactorily met.
The first chapter contains the general introduction whereby the background and justification of the project were presented. In chapter 2 a thrips-resistance and chemical survey of Solanum was conducted. Foliar parts of prominent wild and domesticated tomatoes were challenged with thrips her- bivory and analyzed by untargeted 1H NMR metabolomics. Results showed that thrips resistance was related to acylsugars. These glycolipids are viscous metabolites exudated by the glandular trichomes, specifically by type-4 glands, in Solanum spp. No chemical compounds in the lamella of tomato leaves could be identified in this trial as resistance factors against WFT. In a second experiment (Chapter 3) the survey was extended to other accessions in order to explore in depth the allelochemical potential against WFT of all known trichomal defenses in Solanum. In this part different resistance mechanisms in which mechanical and chemical defenses work coordinately to fend thrips off were observed. In all cases thrips come in contact with exudates from glandular trichomes, mainly of the types 4 and 6. These exudates involve different defense strategies. For instance, acylsugars and sesquiterpenic carboxylic acids, as oily substances, entangle the insects in between the trichomes, preventing thus thrips from reaching the leaf surface to feed on. This resistance mechanism proved itself effective in deterring WFT provided that hairiness and exudate yield were very high. Long-chain methylketones in the exudate, as broad-spectrum insecticides, showed contact toxicity to WFT. However, efficient detoxification mechanisms in WFT limit the effectiveness of these toxins. Thrips recover quickly from the knocking out effect of these ketones once exposure to them ceases. Surprisingly, the most effec- tive and efficient defense mechanism was that observed among the domesticated tomatoes (Solanum lycopersicum). Polyphenol oxidase-catalyzed polymerization of rutin upon rupture of type-6 glandu- lar trichomes on S. lycopersicum literally welds thrips to the leaf surface. When the density of these glands is sufficiently high, like in the yet unidentified cultivar LXX, the insect unavoidably tears a lot of type-6 glands accumulating in consequence an amount of polymers high enough to immobilize it. This defense mechanism, described before in relation to other insect species, worked very well on WFT. In contrast to other defenses, it prevented thrips from feeding on more plants, thereby avoiding
any further virus spread. Cultivar LXX, therefore, represents a promising source of resistance to this pernicious herbivore, which may alleviate the extraordinary pressure currently existing on plant sci- ences to come up with alternatives for crop protection against major pests. In addition, considering that the greatest danger posed by thrips on tomato is the transmission of devastating tospoviruses, such as tomato spotted wilt virus and impatiens necrotic spot virus, this resistance strategy certainly is the ideal defense mechanism for tomato and possibly other crops. The fact that such trait is already prin- ted in the genome of S. lycopersicum makes it a unique and exceptional opportunity to realize thrips resistance in all susceptible cultivars.
The genetics of chemically-based thrips resistance in tomato was also investigated in a further step (Chapter 4). A chromosomal substitution set consisting of 76 introgression lines between S. pen- nellii LA716 and S. lycopersicum var. Moneymaker, was chemotyped by NMR metabolic profiling and screened for thrips resistance. Nine quantitative trait loci for thrips resistance and 268 for foliar metabolites were independently identified. Unfortunately, none of the 26 detected foliar metabolites was associated with thrips resistance.
Another important candidate group of allelochemicals explored in this project was the phenolics, specifically the esters of hydroxycinnamic acids, of which chlorogenic acid (5-caffeoylqui- nic acid) is one of the most widely distributed in the plant kingdom. To evaluate their potential as chemical defenses against WFT and discover possible structure effects on their anti-insect activity a series of nine hydroxycinnamoylquinic acids (HCQA) were isolated from the richest source known in nature, green coffee beans (Chapter 5). The great versatility and preparative capacity of countercur- rent separations, in particular centrifugal partition chromatography, were exploited in this project to isolate the major HCQAs present in green coffee beans, namely caffeoylquinic acid, feruloylquinic acid and dicaffeoylquinic acid. The respective regioisomers of these HCQAs were purified as well.
Because of the significant solubility differences between these metabolites a novel gradient method based on salting-out effects was developed to control the elution of these analytes. The separation of 5-caffeoylquinic acid, 5-feruloylquinic acid, and 3,5-dicaffeoylquinic acid was successfully achieved by means of a salting-out gradient using ethyl acetate-hexane as the stationary phase and an ionic gradient of LiCl as the mobile phase in one case and (NH4)2SO4-KNO3 in another. Despite the greater analytical range and the scaling-up superiority of countercurrent separations compared to other chro- matographic methods these techniques still have not gained wide popularity. Poor automation and laborious method development are characteristic features of countercurrent separations that discou- rage potential new users. Such drawbacks prevent countercurrent separations from competing with predominant liquid chromatography to find their place in every single science laboratory.
In the next and final part of the project the activity of 5-caffeoylquinic acid and its regioisomers, 3-CQA and 4-CQA, against thrips larvae was investigated (Chapter 6). These phenolic metabolites were assayed in artificial diet bioassays at a concentration of 1% with and without polyphenol oxi- dase. Results suggest that the anti-insect properties of CQA extensively reported in the scientific literature may not be connected to its reactive catechol moiety but to its high acidity, in contradic- tion with the main predicted modes of action. This supports the hypothesis that insects, as opposed to pathogens, have different ways to neutralize the adverse physiological effects known to phenolic compounds. The strong influence of pH observed on larval performance also suggests that increased vacuolar accumulation of organic acids, such as chlorogenic acid, in otherwise susceptible foliar tis- sue may contribute to host-plant resistance against herbivores with neutral or alkaline digestive tracts.
Many generalizations are often made about the activity of phenolic compounds. However, the results of these bioassays warn plant scientists about the dangers of such assumptions. As a very diverse class of secondary metabolites phenolics and their role in plant-insect interactions should be evaluated on a case-to-case basis as long as their specific modes of action are not fully elucidated.
Due to the complexity of the chemistry that controls the interaction between organisms as well as their response to abiotic factors, metabolomics, as the most comprehensive analytical method, should be the default tool in life sciences to investigate changes on the metabolic level. The need for holistic approaches in systems biology has made metabolic profiling a standard practice nowadays.
However, metabolomics is still far from meeting its original goal since no analytical platform or com- bination of them can produce a full analysis of the metabolome. Nuclear magnetic resonance for example, albeit offering great throughput, analytical range, elucidation and reproducibility advan- tages unfortunately lacks in sensitivity compared to MS-based technologies. In this project many important primary and secondary foliar metabolites were identified in tomato but those responsible for the observed variation in thrips resistance were in some instances elusive to the 1H NMR analysis.
Because of this relative insensitivity drawback of NMR, an important class of allelochemicals could not be profiled to complete the whole picture of potential defenses against thrips in Solanum, the gly- coalkaloids. Glycoalkaloids, such as tomatine, are very well known effective plant defenses especially abundant in Solanum. Glycoalkaloids have received little attention in the past decades as a promi- sing anti-insect alternative due mainly to their inactivation upon binding with membrane phytosterols.
However, this complex formation that certainly favors chewing insects may not take place in piercing- sucking herbivores like WFT, where alkaloid uptake may occur before sterol binding. The genetic and hence chemical diversity harbored by the numerous wild Solanum species offers a perfect system to conduct structure-activity relationship studies on the effect of this relevant class of allelochemicals on thrips. Glycoalkaloids with a strong negative effect on WFT could join the set of effective resistance traits hereby described to constitute a multi-target defense system whereby a sustainable and stable thrips resistance in the domesticated tomatoes could be realized.
