Dissertations / Theses on the topic 'Plant defenses'

Because organisms have limited resources to allocate to multiple life history traits, the Optimal Defense Theory (ODT) and the Growth-Differentiation Balance Hypothesis (GDBH) were developed by terrestrial plant ecologists to predict intraindividual defense allocation based on the cost of defense and these life history trade-offs. However, these theories have garnered equivocal experimental support over the years and are rarely experimentally extended from predictions of plant physiology to the palatability of the tissues an herbivore experiences. We therefore examined tissue palatability, nutritional value, and defense mechanisms in multiple Dictyotalean seaweeds in two Caribbean locations, using two herbivores. Relative palatability of tissues varied greatly with algal species, grazer species, and location. Because older bases were not consistently defended, GDBH did not predict relative palatability. We could not reject ODT without intensive measures of tissue fitness value and herbivore risk, and this theory was therefore not useful in making broad predictions of tissue palatability. In testing the physiological predictions of these theories, we found the young, growing apices of these seaweeds to be generally more nutritionally valuable than the old, anchoring bases and found organic-rich apices to be more chemically deterrent, thus supporting ODT. However, the combined chemical, nutritional, and structural traits of these algae all influenced herbivore choice. As a result, these patterns of apical value and chemical defense reflected palatability of live tissues for only one of five algal species, which rendered ODT and GDBH poor predictors of relative palatability for most algae.

Induction of phenylpropanoid metabolism in many plants is associated with the induction of plant defence responses. Among these are the accumulation of phenylpropanoid-derived phytoalexins, increase in lignification around infected sites, and the accumulation of wall-bound phenolic compounds. I show in this work, that H11 hybrid cell suspension cultures when treated with either of three elicitors respond with an increase in phenylpropanoid metabolism. Activation proceeds rapidly from PAL and 4CL mRNA accumulation, to a massive increase in extractable PAL enzyme activity and finally there is accumulation of specific phenolic compounds in the cell extracts, culture filtrates, and cell walls. In addition, elicitor treatment causes cells to turn brown, indicative of phenolic compound accumulation. As in other plants, induction is dependent on culture age, is dose dependent, and the kinetics of induction is the same with all three elicitors. Based on the previously established mode of action of PGA lyase as an elicitor, it is concluded that in poplar, as in other plants, defence responses can be induced by elicitors from both fungal and plant cell wall origin. These results illustrate the successful use of plant suspension cultures as a simplified system to study inducible defence responses. In addition, and consistent with the ubiquitous nature of phenolics in poplar, phenylpropanoid metabolism may play an important role in plant defence responses in this species.
Science, Faculty of
Botany, Department of
Graduate

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.

[Truncated abstract] A range of biotic and abiotic stresses increase levels of reactive oxygen species (ROS) in plants due to perturbations of chloroplast and mitochondrial metabolism and the generation of ROS in defence responses. The polyunsaturated fatty acids of membrane lipids are susceptible to ROS induced peroxidation yielding various aldehydes, alkenals and hydroxyalkenals including the cytotoxic compound 4-hydroxy- 2-nonenal (HNE). HNE has the potential to cause substantial oxidative damage in cells via its reactivity with sulfhydryl groups of cysteine (Cys) and lipoic acid, the imidazole group of histidine (His) and the ?-amino group of lysine (Lys) protein residues. Analysis of the components of the plant respiratory electron transport chain to HNE revealed a particular susceptibility to inhibition of activity of the alternative oxidase (Aox). Incubation with HNE prevented dimerisation of Aox protein, suggesting that one site of modification was the conserved cysteine residue involved in dimerisation and activation of this enzyme (Cys1). However, a naturally occurring isoform of Aox lacking Cys1 and unable to dimerise, LeAox1b from tomato, was equally sensitive to HNE inhibition, showing that other amino acid residues in Aox also interact with HNE and are likely responsible for inactivation of the enzyme. ... The broader impact of HNE on the whole Arabidopsis mitochondrial proteome was examined by use of various 2-dimensional gel separation techniques coupled with use of HNE-adduct antibodies. 32 proteins involved in a number of mitochondrial functions were found to be susceptible to modification by HNE, including components of the electron transport chain, the TCA cycle, as well as proteins involved amino acid metabolism and stress-responses. Implications of modification of these proteins by HNE are discussed. As HNE is produced in vivo during oxidative stress, the profile of mitochondrial targets of HNE was examined from Arabidopsis cell cultures exposed to various oxidative stress inducers. Menadione and hydrogen peroxide induced oxidative stress throughout the cell, while antimycin A initiated a mitochondrial targeted stress. A differential profile of mitochondrial proteins was observed to be modified by HNE in the various treatments. These results also showed that induction of stress within a whole cell can impact lipid peroxidation within the mitochondria. Overall, this work showed the presence and production of HNE in plant cells, and that HNE, both exogenous and endogenous, has the ability to modify a specific subset of mitochondrial proteins. In several cases this HNE modification was shown to have functional or structural consequences.

As sessile organisms, plants evolved a plethora of defenses against their attackers. Given the role of plants as a primary food source for many organisms, plant defense has important implications for community ecology. Surprisingly, despite the potential to alter entire food webs and communities, the factors determining plant investment in defense are not well-understood, and are even less understood considering the numerous symbiotic interactions in the same plant. Legume-rhizobia symbioses engineer ecosystems by fixing nitrogen from the atmosphere in trade for plant photosynthates, yet connecting symbiotic resource exchange to food web interactions has yet to be established. Here I test how rhizobia influence plant defense and tritrophic interactions in lima bean (Fabaceae - Phaseolus lunatus L.): a model plant in chemical ecology research characterized by a broad range of different defenses. Examining suites of traits among lima bean genotypes, highly cyanogenic cultivars and wild type plants (high cyanotypes) produce more hook-shaped trichomes, as a putative combined approach of chemical and mechanical defenses, forming defense syndromes to protect against multiple feeding guilds (Chapter 2). Testing costs that may have contributed to forming tradeoffs among strategies, high cyanotypes show reduced fitness under plant-plant competition relative to low cyanotypes, but when challenged with herbivory, high cyanotypes fitness reductions are no longer evident (Chapter 3). Young leaves, not reproductive organs, are the most cyanogenic lima bean organ, and removal quantitatively decreases fitness, supporting assumptions that the most valuable tissues will be most highly defended (Chapter 4). Testing the degree to which nitrogen-fixing rhizobia contribute to cyanogenesis, high cyanotypes form more nodules than low cyanotypes. Quantitative relationships between nodule number and plant traits highlight the role symbiotic investment plays a role in plant defense and nutritive phenotype, while simultaneously, genotypically-determined levels of defense shape plant investment in symbiosis (Chapter 5). Interestingly, traits that trade off by cyanotype (i.e. high cyanogenesis but low indirect defense) reflect the patterns in plants with nitrogen-fixing rhizobia. Rhizobia-inoculated lima beans show reduced indirect defenses, recruiting fewer parasitoid wasps (Chapter 6) and predatory ants (Chapter 7). Examining plant-ant attraction in greater detail, ants prefer headspace regions above EFN droplets, corresponding with species-specific differences in suites of volatiles, indicating EFN, like floral nectar, can be scented to manipulate insect behavior (Chapter 8). Overall, understanding when investing in traits to recruit predators is more effective than investing in defensive chemistry, and how particular ecological contexts, such as symbioses can influence the outcome of defense allocation strategies remains a fascinating area of research. Determining the mechanisms underlying why rhizobia and other belowground microbial symbionts influence their host plants' above ground interactions, whether plants traits affected by symbiotic microbes are simply a function of the costs and benefits from resource exchange, or whether symbionts can influence the success of primarily direct versus indirectly defended plants is an important question for understanding complex trophic systems and connecting to agricultural implications for more effective biological pest control.

Plants synthesize a diverse array of phytochemicals in response to interactions with herbivores, pathogens, and commensal microbes. These phytochemicals may simultaneously enhance crop defense and quality, representing a potential pest management strategy. However, plant chemical responses to different types and levels of biotic interactions remain unclear, particularly in fruit tissues, and the feasibility of inducing these defenses through elicitor application in field environments also requires further examination. Thus, apples were used to 1) examine the impact of distinct communities of biotic interactions among plants, insects, and microbes on fruit phenolic chemistry, and 2) examine the impact of the phytohormones jasmonic acid (JA), salicylic acid (SA), and melatonin (M) on fruit phenolic chemistry and resistance against pests and pathogens. Ultimately, phenolic defenses were induced by fungal damage primarily in ripe pulp tissues, where there was also a positive relationship between fungal endophyte and phenolic diversity, supporting a broad hypothesis that chemical diversity may increase with biotic diversity. Additionally, two compounds were upregulated in response to fungal damage: chlorogenic acid and an unidentified benzoic acid. Elicitor applications did not affect phenolic chemistry, but the combined application of JA-SA analogues had some chemical or physical effect, as this treatment reduced emergence of the insect Rhagoletis pomonella. Thus, fruit induced defenses may be tissue-specific and subject to temporal, environmental, or genotypic variation. Overall, these chapters examined the relationship between biotic interactions and induced fruit chemistry, with the goal of improving understanding of plant-microbe-insect interactions and incorporating these interactions into more sustainable agricultural practices.
Master of Science
Plants may produce a diverse array of defensive phytochemical compounds in response to interactions with herbivores, pathogens, and the microorganisms that reside within plant tissues. These phytochemicals may simultaneously improve crop defenses and quality, representing a potential agricultural management strategy. However, plant chemical responses to different types and levels of biotic interactions are not well-understood, particularly in fruit tissues, and the feasibility of activating these defenses in fruits through the application of phytohormones that regulate defense pathways as a potential management strategy also requires further examination. Thus, apples were used to 1) examine the impact of distinct communities of biotic interactions among plants, insects, and microbes on fruit chemistry, focusing on phenolics, an important class of phytochemical compounds, and 2) examine the impact of the defense-activating phytohormones jasmonic acid (JA), salicylic acid (SA), and melatonin (M) on fruit phenolic chemistry and resistance against pests and pathogens. Ultimately, phenolic defenses were activated by fungal damage primarily in ripe pulp tissues, where there was also a positive relationship between fungal endophyte and phenolic diversity, supporting a broad hypothesis that chemical diversity may increase with biotic diversity. Additionally, two compounds were produced in response to fungal damage: chlorogenic acid and an unidentified benzoic acid. Phytohormone applications did not affect phenolic chemistry, but the application of the combined JA-SA analogues had some chemical or physical effect, as this treatment reduced emergence of the insect Rhagoletis pomonella. Overall, the phytochemical defenses activated by biotic interactions in fruits may occur primarily in certain tissue types, and may also vary due to environmental conditions, time of year, or plant species. These chapters examined the relationship between fruit chemistry and biotic interactions with the goal of improving understanding of plant-microbe-insect interactions and incorporating these interactions into more sustainable agricultural practices.

Plants produce a large number of specialized or secondary compounds that aid in their reproduction and protection against biotic and abiotic stress. In this work I investigated the metabolism and function of terpenes, the largest class of specialized metabolites, in switchgrass and carrot. Switchgrass (Panicum virgatum L.), a perennial C4 grass of the Tallgrass Prairie, represents an important species in natural and anthropogenic grasslands of North America. Its natural resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. I have investigated the metabolism of terpenes in switchgrass leaves and roots in response to herbivory or defense hormone treatments and the application of drought. With a focus on volatile terpene metabolites, I functionally characterized over thirty genes (terpene synthases, TPSs), of which one third could be correlated with the production and release of volatile monoterpenes and sesquiterpenes that likely function in direct chemical defense or in the attraction of insect predators or parasitoids. Drought stress application caused switchgrass roots to accumulate a larger amount of oxygenated terpenes and presumably non-volatile terpenes, the function of which in direct or indirect drought stress protection requires further investigation. I also examined the metabolic dynamics and role of the monoterpene borneol, which accumulates at high concentrations in the roots of switchgrass and to a lower extent in the roots of the close relative Setaria viridis, in root microbe interactions. Although we demonstrated a successful RNAi based knock down of the borneol terpene synthase TPS04, we found no immediate evidence that borneol significantly modifies bacterial communities in the root. Further studies on Setaria and equivalent RNAi lines in switchgrass will provide more detailed and needed insight to decipher the role of monoterpene accumulation in grasses interactions with mutualists, pathogens, and pests. In an applied project, I investigated terpene specialized metabolism in carrot (Daucus carota L.) to identify genetic determinants of carrot aroma and flavor. To determine central enzymes which contribute to the terpene component of carrot volatile blends, we first analyzed tissue specific expression patterns of carrot terpene synthase genes (TPS) in the genomic model carrot (cv. DH1) and in roots of four aromatically unique colored carrot genotypes (orange-4943B, red-R6637, yellow-Y9244A and purple-P7262). We selected nineteen key biosynthetic enzymes involved in terpene formation and compared in vitro products from recombinant proteins with native volatile profiles obtained from DH1 and colored carrot genotypes. We biochemically characterized several highly expressed TPSs with direct correlations to major compounds of carrot flavor and aroma including germacrene-D (DcTPS11), (DcTPS30) and -terpinolene (DcTPS03). Random forest analysis of colored carrot volatiles revealed that nine terpene compounds are sufficient for distinguishing the flavor and aroma of raw colored carrots. Interestingly, accumulation of specific terpene compounds rather than chemical diversity is responsible for differences in sensory quality traits in colored genotypes. As accumulations of specific terpene compounds can contribute to the undesired flavor in carrot, our report provides a detailed roadmap for future breeding efforts to enhance carrot flavor and aroma.
Doctor of Philosophy

Small RNAs are important regulators for a variety of biological processes, including leaf development, flowering-time, embryogenesis and defense responses. Most ancient miRNAs are conserved among different plant species and well characterized, while young MIRNA genes are considered to be non-conserved, highly species-specific and less well-studied. miR163 is a non-conserved miRNA and its locus has evolved recently by inverted duplication events of its target gene. Previously, we have shown that miR163 acts as a negative regulator of defense response. However, it remains unclear how miR163 and its targets are being regulated in response to pathogen attacks. Here, we further elucidated the molecular controls and the involvement of miR163 and its targets in plant defense response. Elevated level of miR163 was observed by Pst treatment in Arabidopsis thaliana, and this upregulation was found to be important in controlling the accumulation of its targets (PXMT1 and FAMT), to which they were also inducible by Pst treatment. Transcript and protein level analyses in transgenic plants overexpressing miR163-resistant form of PXMT1 or FAMT provided evidence for miR163 in fine-tuning its targets, suggesting that the stress-inducible miR163 and its targets act in concert in affecting defense genes expression. Epigenetically, histone deacetylation was found to involve in the repression of miR163 targets before and after Pst infection. Our findings revealed additional mechanistic insights to the controls and the evolutionary significance of young miRNA in mediating plant defense pathways against biotic stresses.

Dissertation (PhD)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses.
AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.

In this thesis, I combined field, greenhouse and common-garden experiments to examine the ecological and evolutionary consequences of plant-herbivore interactions and the genetic architecture of fitness-related traits in the insect-pollinated, self-incompatible, perennial herb Arabidopsis lyrata. More specifically, I examined (1) whether damage to leaves and inflorescences affects plant fitness non-additively, (2) whether trichome production is associated with a cost in terms of reduced tolerance to leaf and inflorescence damage, (3) whether young plant resistance to a specialist insect herbivore varies among populations, and (4) whether the evolution of flowering time, floral display and rosette size is constrained by lack of genetic variation or by genetic correlations among traits. A two-year field experiment in a Swedish population showed that damage to rosette leaves and to inflorescences can affect both current and future plant performance of A. lyrata, and that effects on some fitness components are non-additive. A two-year field experiment in another Swedish population indicated that trichome-producing plants are not less tolerant than glabrous plants to leaf and inflorescence damage. In a greenhouse experiment, acceptability of young plants (5-6 weeks old) to ovipositing females and damage received by Plutella xylostella larvae varied considerably among twelve A. lyrata populations. Both oviposition and leaf damage were positively correlated with rosette size, but trichome density in the trichome-producing morph was apparently too low at this developmental stage to influence resistance to P. xylostella. In a common-garden experiment, flowering time, floral display and rosette size varied among four Scandinavian A. lyrata populations, and displayed significant additive genetic variation in some populations. Yet, strong genetic correlations between flowering start and number of flowers, and between petal length and petal width suggest that these traits may not evolve independently. Taken together, the results indicate the need to consider possible long-term and non-additive effects of herbivore damage to different plant parts, that there is no trade-off between trichome production and tolerance to herbivory, that the importance of morphological defenses against herbivory may change through plant ontogeny, and that considerable genetic variation for traits such as flowering time and floral display can be maintained in natural plant populations.

Orientador: Maria Lígia Rodrigues Macedo
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-25T23:17:16Z (GMT). No. of bitstreams: 1 Bezerra_CezardaSilva_M.pdf: 16272882 bytes, checksum: c9bdf2bf628fb555d1bc1ccc47c972c5 (MD5) Previous issue date: 2014
Resumo: Os inibidores de proteases de plantas têm sido amplamente estudados como uma alternativa para o controle de insetos-praga devido à capacidade de inibir enzimas envolvidas na digestão. Assim sendo, o objetivo deste trabalho foi estudar a atividade biológica do inibidor de tripsina de sementes de Inga vera (IVTI) sobre o desenvolvimento, fisiologia nutricional e atividade enzimática de Anagasta kuehniellla. Larvas neonatas (n=40) foram mantidas em dieta artificial sem inibidor (controle) ou contendo 1% de IVTI (p/p) até atingirem o quarto e quinto instares. As análises realizadas determinaram o efeito sobre o desenvolvimento destas, desde período larval até emergência dos adultos. Através de outras aferições elaboramos uma tabela de parâmetros nutricionais. A atividade proteolítica do homogenato intestinal e fecal foi analisada através de zimograma e ensaios enzimáticos in vitro, utilizando BApNA e Suc-AAPF-pNA como substratos para tripsina e quimotripsina, respectivamente. IVTI foi incubado com o homogenato intestinal larval para verificar a degradação do mesmo. O consumo de IVTI pelas larvas provocou uma redução de cerca de 50% no peso médio larval e uma redução significativa taxa de sobrevivência de 15%, bem como o prolongamento do período larval em 8 dias. A análise dos índices nutricionais revelou uma redução na eficiência de conversão do alimento ingerido e digerido e um aumento no custo metabólico, sugerindo que IVTI apresenta efeito antinutricional para esta espécie. IVTI alterou a atividade proteolítica intestinal das larvas com a redução atividade tríptica e o aumento da atividade quimotríptica. Na análise fecal, os níveis da atividade tríptica foram semelhantes tanto nas fezes das larvas alimentadas em dieta controle quanto contendo inibidor, entretanto foi observado um aumento no nível da atividade quimotríptica nas larvas alimentadas com inibidor. IVTI não foi degradado pelas enzimas intestinais, sendo excretado nas fezes e permanecendo com sua atividade inibitória ativa. O zimograma não revelou nenhuma forma variante de enzima nas larvas alimentadas com inibidor, mas foi possível observar quais sofreram influências pelo mesmo. Com base nesses resultados, IVTI apresentou uma atividade tóxica e antinutricional contra A. kuehniella
Abstract: Plant protease inhibitors have been extensively studied as an alternative for the control of insect pests because of their ability to inhibit enzymes digestive enzymes. In this work, the biological activity of trypsin inhibitor of Inga vera seed (IVTI) on the development, nutritional physiology and enzyme activity of Anagasta kuehniellla was evaluated. Neonate larvae (n=40) were maintained on artificial diet without inhibitor (control) or containing 1% IVTI (w / w) until fourth and fifth instar. This bioassay determined the effect on the development from the larval period to adult emergence. Through other measurements prepared a table of nutritional parameters. The proteolytic activity of intestinal and fecal homogenate was analyzed by zymography and enzymatic assays, in vitro, using BApNA and Suc-AAPF-pNA as substrates for trypsin and chymotrypsin, respectively. IVTI was incubated with the larval gut and check the degradation of the inhibitor. IVTI consumption by the larvae resulted in a reduction of about 50% of larval weight and a significant larval survival rate of 15%, as well as the extension of the larval period to 8 days. Nutritional analyses showed a reduction of efficiency of conversion of food eaten and digested and an increase in metabolic cost, suggesting that IVTI produces an anti-nutritional effect for this specie. IVTI changed the proteolytic activity in the gut of the larvae with decrease of trypsin activity and increase of chymotrypsin activity. Fecal analyses, the levels of trypsin activity were similar in the feces of larvae fed on control diet as containing inhibitor, however there was an increase in the level of chymotrypsin activity in larvae fed with inhibitor. IVTI was not degraded by intestinal enzymes, but it excreted in the feces and their inhibitory activity remained active. Zymogram revealed no variant form of the enzyme in the larvae fed inhibitor, but was observed which were influenced by it. Based on these results, IVTI presented an anti-nutritional and toxic activity against A. kuehniella
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

Chemically mediated interactions have important ecological and evolutionary effects on populations and communities. Despite recognition that herbivory can significantly affect the biomass and composition of freshwater macrophyte communities, there are few investigations of chemical defenses among freshwater vascular plants and mosses and none of freshwater red algae. This study compares the palatability of five species of freshwater red algae (Batrachospermum helminthosum, Boldia erythrosiphon, Kumanoa sp., Paralemanea annulata, and Tuomeya americana) that occur in the southeastern United States relative to two co-occurring macrophytes (the chemically defended aquatic moss Fontinalis novae-angliae and the broadly palatable green alga Cladophora glomerata). We assessed the potential role of structural, nutritional, and chemical traits in reducing macrophyte susceptibility to generalist crayfish grazers. Both native and non-native crayfish significantly preferred the green alga C. glomerata over four of the five species of red algae. B. erythrosiphon was palatable, while the cartilaginous structure of P. annulata reduced its susceptibility to grazing, and chemical defenses of B. helminthosum, Kumanoa sp., and T. americana rendered these species as unpalatable as the moss F. novae-angliae. Extracts from these latter species reduced feeding by ~30-60% relative to solvent controls if tissues were crushed (simulating herbivore damage) prior to extraction in organic solvents. However, if algae were first soaked in organic solvents that inhibit enzymatic activity and then crushed, crude extracts stimulated or had no effect on herbivory. B. helminthosum, Kumanoa sp., and T. americana all exhibited "activated" chemical defenses in which anti-herbivore compounds are produced rapidly upon herbivore attack via enzymatic processes. In an additional accept/reject behavioral assay, B. helminthosum extracts reduced the number of crayfish willing to feed by >90%. Given that three of the five red algal taxa examined in this study yielded deterrent crude extracts, selection for defensive chemistry in freshwater rhodophytes appears to be substantial. Activated chemical defenses are thought to be an adaptation to reduce the resource allocation and ecological costs of defense. As such, activated chemical defenses may be favored in freshwater red algae, whose short-lived gametophytes must grow and reproduce rapidly. Roughly 20% of the known chemical defenses produced by marine algae are activated; further examination is needed to determine whether the frequency of activated chemistry is higher in freshwater red algae compared to their marine counterparts. Continued investigation of chemical defenses in freshwater red algae will contribute to among-system comparisons, providing new insights in the generality of plant-herbivore interactions and their evolution.

Las plantas, debido a que no pueden desplazarse cuando el ambiente no les es favorable, han desarrollado una inmunidad innata que les permite ser resistentes a casi todos los microbios e insectos. El priming de las defensas basales hace que la respuesta de la planta sea más temprana y más intensa si un patógeno virulento intenta colonizar la planta. Además de los mecanismos que se proponen para explicar el priming de las defensas basales, se señala otro nuevo, que es la necesidad de la homeostasis en el control de las especies reactivas de oxígeno (ROS). El priming por BABA (ácido beta-aminobutírico) genera un ambiente celular más oxidado que necesitará estar estrechamente controlado para no provocar daños en la célula. Por otra parte, cuando los mecanismos primarios de priming están bloqueados, como el priming de ROS o de calosa, BABA es capaz de actuar induciendo líneas de defensa más tardías o secundarias, indicando que el priming de las defensas es un mecanismo multicomponente. Así mismo, se demuestra que las condiciones ambientales pueden influir de forma decisiva en el resultado final de una interacción planta-patógeno, pudiendo pasar de generar resistencia a susceptibilidad. Mediante análisis metabolómico usando UPLC-qTOF analysis se observa que la ruta más importante en el priming contra el hongo Plectosphaerella cucumerina es la ruta del triptófano, y dirigiéndose hacia una rama de la ruta distinta a los indolglucosinolatos, camalexina o IAA.

For decades scientists believed that herbivory had minimal impact on freshwater ecosystems. We now know that herbivory in freshwater systems equals or exceeds herbivory in terrestrial and marine systems. In extreme cases, herbivores can change clear, macrophyte dominated ecosystems into turbid plankton dominated ecosystems. Even though research on plant-herbivore interactions in freshwater systems has increased, there is still much that is unknown. This thesis is comprised of four studies investigating freshwater plant-herbivore interactions across multiple spatial scales. The first study investigated how induced chemical defenses in Cabomba caroliniana suppress herbivore consumption and growth as well as how this herbivore-generated change in plant chemistry affects the growth of plant associated microbes. At the spatial scale of individual ponds or lakes, consumers that induce their host plants may also be indirectly affecting other consumers and microbial pathogens via changes in this shared resource. The second study moves to an ecosystem scale and investigates how exotic versus native apple snails may impact Everglades' habitats. We investigated plant preference, consumption, growth and conversion efficiencies in the singly native apple snail to occur in the U.S. (Pomacea paludosa) versus four introduced species (P. canaliculata, P. insularum, P. haustrum and P. diffusa). We found that even though plant preferences are similar, invasive snails tend to eat more, grow more rapidly, and sometimes more efficiently than natives. This suggests that invasive species could have a large impact on the environment, especially the abundance of submerged plants. The third study investigated how palatability of freshwater plants varies with latitude (i.e. geographic scale). Increased herbivory at lower latitudes is hypothesized to select for increased plant defenses, which has been shown to be true for tropical forests, salt marshes, and seaweeds. When we contrasted eight confamilial plants collected in Indiana versus Southern Florida, three of four herbivores significantly preferred northern plants. When we evaluated a second set of plants collected from Indiana versus Central Florida, only one of three herbivores preferred the northern plants. Overall, our results suggest a preference for northern plants, but the strength of this relationship was variable. We hypothesize that this variability may be driven by 1) local variance in herbivore pressure that creates variance in plant defenses, and/or 2) the effect of winter length on the survival and feeding rate of herbivores. The final study expanded to a world scale, and investigated herbivore preference for native vs exotic plants. We found that both N. American crayfish and S. American snails preferred exotic plants over confamilial natives, despite responding to different plant characteristics. The single species of apple snail that occurs in N. American showed no preference for native or exotic plants from a N. American perspective, but instead exhibited preferences that correlated with its history of evolution in S. America. As the N. American species is a sister species of the S. American snails, feeding by the N. American snail appears more affected by its S. American lineage than its recent history in N. America. This suggests that phylogenetic legacy will affect choices of the herbivore as well as resistance or susceptibility of plants.

Invasive exotic species threaten native biodiversity, alter ecosystem structure and function, and annually cost over $100 billion in the US alone. Determining the ecological traits and interactions that affect invasion success are thus critical for predicting, preventing, and mitigating the negative effects of biological invasions. Native herbivores are widely assumed to facilitate exotic plant invasions by preferentially consuming native plants and avoiding exotic plants. Here, I use freshwater plant communities scattered broadly across the Southeastern U.S. to show that herbivory is an important force driving the ecology and evolution of freshwater systems. However, native consumers often preferentially consume rather than avoid exotic over native plants. Analyses of 3 terrestrial datasets showed similar patterns, with native herbivores generally preferring exotic plants. Thus, exotic plants appear defensively nave against these evolutionarily novel consumers, and exotic plants may escape their coevolved, specialist herbivores only to be preferentially consumed by the native generalist herbivores in their new ranges. In further support of this hypothesis, a meta-analysis of 71 manipulative field studies including over 100 exotic plant species and 400 native plant species from terrestrial, aquatic, and marine systems revealed that native herbivores strongly suppressed exotic plants, while exotic herbivores enhanced the abundance and species richness of exotic plants by suppressing native plants. Both outcomes are consistent with the hypothesis that prey are susceptible to evolutionarily novel consumers. Thus, native herbivores provide biotic resistance to plant invasions, but the widespread replacement of native with exotic herbivores eliminates this ecosystem service, facilitates plant invasions, and triggers an invasional meltdown. Consequently, rather than thriving because they escape their co-evolved specialist herbivores, exotic plants may thrive because their co-evolved generalist herbivores have stronger negative effects on evolutionarily nave, native plants.

The water hyacinth (Eichhornia crassipes (Mart.) Solms-Laub (Pontederiaceae)) biological control programme makes use of tight plant-insect interactions to control the weed, by reestablishing the interactions between the plant and its natural enemies. Since the beginning of the water hyacinth biological control initiative, the impact of biological control agent herbivory on water hyacinth’s population growth and fitness have been well documented; however, very few investigations have been conducted to determine whether herbivory elicits insect-induced responses by water hyacinth. Studies were conducted to determine the presence and function of water hyacinth insectinduced responses, using the plant activator, BION®, in attempt to determine the plant hormone-mediated pathways regulating the final expressions of insect-induced defences in response to herbivory by the phloem-feeder, Eccritotarsus catarinensis (Carvalho) (Hemiptera: Miridae) and the leaf chewer, Neochetina bruchi Hustache (Coleoptera: Curculionidae). BION® (Syngenta, acibensolar-S-methyl (benzothiadiazole)) is a dissolvable, granular formulation that contains a chemical analogue of the plant hormone, salicylic acid (SA), which typically regulates defences against pathogens. The application of BION® results in the induction of the SA-mediated defence pathways in plants (activation of defences against pathogens), and consequently the inhibition of the jasmonic acid (JA)- mediated defence pathways (de-activation of defences against insect herbivores). To test for induced defence responses in water hyacinth, plants treated with BION® and then subjected to herbivory, were compared to un-treated plants that were also subjected to herbivory, BION®-only treated plants and control plants. The application of BION® did not confer resistance against the two insect herbivores, as herbivory, reductions in chlorophyll content and plant growth (leaf production and second petiole lengths) significantly increased in comparison to non-BION® treated plants. Furthermore, palatability indices significantly increased (>1.00) in BION® treated plants, reflecting increased weevil preferences for SAinduced water hyacinth plants. This concluded that SA-mediated defences are not effective against insect herbivory in water hyacinth plants, but are in fact palatable to insect herbivores, which reflects ecological and physiological costs of SA-mediated defences (pathogen defences) in water hyacinth. Biochemical analyses of leaves exhibited increases in nitrogen content in BION® treated plants. These elevated levels of nitrogenous compounds account for the increases in mirid and weevil preferences for BION® treated plants. The increases in nitrogenous compounds are probably structural proteins (e.g. peroxidises), because leaves treated with BION® increased in toughness, but only when exposed to herbivory. Regardless, insect herbivory was elevated on these leaves, probably because the nitrogenous compounds were nutritionally viable for the insects.

Plant-herbivore interactions play a significant role in the structure and functioning of ecosystems. Co-evolutionary theory suggests that plant defenses evolved due to herbivores and herbivore pressure can shape the genetic composition of their food resources. We used interactions between North American porcupines (Erethizon dorsatum) and trembling aspens ( Populus tremuloides) as a system to investigate this theory's important assumption that herbivores select food sources based on genetically controlled traits. We confirmed that porcupines exhibit intra-specific food selection and that this is linked to the genetic composition of the aspens. We also demonstrated that variation in phenolic glycosides and condensed tannins are strong components of this selection, thereby creating an important link between genetics, plant chemistry, and mammalian herbivory. We investigated potential impacts of porcupine herbivory on aspen using fluctuating asymmetry, however we did not detect any stress on heavily eaten trees, thereby questioning the validity of this tool for this study system.

Las plantas han desarrollado mecanismos sofisticados de defensa para protegerse de los organismos perjudiciales de su entorno. La resistencia depende de múltiples mecanismos de protección que comprenden, tanto barreras físicas y químicas constitutivas, como respuestas inducibles. Estas son activadas ante el reconocimiento de la presencia del patógeno que desencadena en la planta una serie de reacciones defensivas que incluyen una reprogramación transcripcional de los genes de defensa, la producción de metabolitos secundarios (fitoalexinas), el reforzamiento de la pared celular y en algunos casos la muerte celular programada, con el fin de evitar la propagación del patógeno. Uno de los tópicos más importante de investigación en plantas y al cual se están dedicando grandes esfuerzos es entender los procesos de señalización que llevan a la activación de las respuestas de defensa en las plantas, por su particular relevancia en la protección de cultivos de todo del mundo. Las reacciones de defensa tempranas desencadenadas por la percepción del patógeno en las células vegetales comprenden la despolarización de la membrana plasmática, cambios en los niveles de calcio intracelular, la producción de especies reactivas de oxígeno (ROS), seguidos por la activación de proteínas quinasas y los consiguientes procesos de fosforilación reversible de proteínas reguladas por calcio. Las plantas poseen diversas clases de proteínas sensoras de calcio, entre ellas las proteínas quinasas dependientes de calcio (CPKs o CDPKs). La peculiaridad de estas proteínas reside en que en la misma cadena polipeptídica encontramos el dominio quinasa, un dominio autorregulador y el dominio calmodulina de unión a Ca2+, pudiendo ser activadas directamente por la unión del Ca2+. Esta estructura tan particular de las CPKs las convierte en sensores de cambios en los niveles de calcio y transductores en actividad quinasa de proteínas que median los procesos de señalización posteriores. El trabajo de este proyecto de tesis doctoral ha estado dirigido al estudio y la caracterización de la participación de una proteína CPK de la planta modelo Arabidopsis thaliana, la proteína AtCPK1, en la defensa frente a patógenos. Al inicio del trabajo, se disponía de evidencias preliminares obtenidas en el grupo de investigación que demostraban la implicación funcional de esta proteína AtCPK1 en la resistencia de la planta a infección por patógenos, lo que justificaba el interés de este estudio. Los estudios desarrollados en este trabajo demuestran que la expresión del gen AtCPK1 se induce rápidamente en respuesta a la infección por el hongo patógeno Fusarium oxysporum y al tratamiento con elicitores derivados del mismo hongo en plantas de Arabidopsis. Además de la regulación transcripcional, se ha mostrado que la correspondiente proteína está regulada postraduccionalmente. Así, los niveles de acumulación de la proteína AtCPK1 están controlados por la vía de degradación de proteínas dependiente del proteasoma. También se ha observado que la proteína AtCPK1 presenta distintos estados de fosforilación, y en respuesta a la infección fúngica la proteína se fosforila bien por autofosforilación, bien por otras quinasas pendientes de indentificación. Por otra parte, la presencia de iones calcio determina de manera importante la capacidad de interacción de AtCPK1 con otras proteínas, mimetizando el efecto de la infección en relación a las proteínas que interaccionan con AtCPK1. Con el objetivo de entender mejor la participación de AtCPK1 en la respuesta de defensa de la planta y de identificar otros componentes implicados en las rutas de transducción de señales activadas por infección, en este trabajo se ha caracterizado el interactoma de AtCPK1 mediante diferentes estrategias complementarias. Entre las proteínas identificadas que interaccionan con AtCPK1 o asociadas a los complejos multiproteicos en los que participa AtCPK1 se encuentran proteínas de la familia de las 14-3-3, enzimas implicadas en la protección frente a estrés oxidativo (ascorbato peroxidasa y catalasas) y en la detoxificación celular (nitrilasas), así como también proteínas cloroplásticas que participan en el ciclo oxidativo del cloroplasto (la ATP sintasa y proteínas de unión a las clorofilas). Estos estudios además han mostrado una localización cloroplástica de la proteína AtCPK1, añadiendo un nivel de complejidad más en cuanto al compartimento subcelular en el cual AtCPK1 desempeña su función y se añade a las localizaciones anteriormente descritas para esta proteína en peroxisomas y cuerpos lipídicos de las células de Arabidopsis.

Plant Defensins type 1 (PDF1) sont principalement décrites pour leur rôle dans l'immunité innée en réponse à des attaques pathogènes via l'activation de la voie de signalisation de l'éthylène (Et) et de l'acide jasmonique (JA). Les défensines PDF1 du genre Arabidopsis sont également impliquées dans la tolérance cellulaire au zinc chez la levure. In planta, de nombreux résultats mettent en évidence une corrélation entre la forte accumulation des transcrits AhPDF1 et leur contribution dans la tolérance à un excès de zinc. Dans cette étude, l'analyse du transcriptome (qRT-PCR) révèle que les paralogues PDF1s, aussi bien chez A. thaliana que chez A. halleri sont très peu voire pas du tout sensibles au zinc. Toutefois, il y a une spécialisation des PDF1s en réponse à l'activation de la voie de l'acide jasmonique dans le genre Arabidopsis. De plus, la contribution fonctionnelle des membres de la famille PDF1s dans la tolérance au zinc a été caractérisée chez A. thaliana à l'aide d'une approche génétique combinant des mutants KO après insertion d'un ADN-T et la technologie de miRNA artificiel. L'étude de ces mutants souligne par ailleurs la diversité fonctionnelle au sein de la famille des défensines AtPDF1s qui ne confèrent pas toutes la tolérance au zinc. En effet, une diversité de déterminants moléculaires des PDF1s a été mise en évidence lors de cette étude. La forte accumulation des PDF1s n'est pas l'unique paramètre requis pour la tolérance au zinc. Il faut également considérer la spécificité de tissu où s'expriment ces PDF1s. A ces considérations s'ajoutent aussi des régulations post-transcriptionnelles et post-traductionnelles. L'étude de ces modifications est envisagée afin de comprendre la contribution des différentes défensines PDF1s dans la tolérance au zinc
Plant Defensin type 1 (PDF1s) are mainly recognized for their response to pathogen attack via ethylene (Et)/jasmonate (JA) signaling activation pathway. However, PDF1s originating from Arabidopsis genus also showed their capacity to induce cellular zinc tolerance up on expression in yeast. In planta, a group of observation highlighted the correlation of AhPDF1 high transcript accumulation for their contribution to zinc tolerance. Here, transcriptomic analysis (qRT-PCR) revealed that in both A. thaliana and A. halleri species, PDF1 paralogues were barely or not at all responsive to zinc. Nevertheless, there is a species specialization of PDF1s in response to activation of JA-signaling in Arabidopsis genus. In addition, in A. thaliana, the functional contribution of PDF1 members in zinc tolerance was investigated through genetic approach. Examining combination of T-DNA insertion knockout mutant and artificial miRNA, these studies were first direct demonstration of the functional involvement of AtPDF1s in zinc tolerance. These also highlighted the functional diversity among AtPDF1s because not all of them could play a role in zinc tolerance. Indeed, a diversity of PDF1 molecular determinants for zinc tolerance in plants was underlined. Remarkably, PDF1 high transcript is not the only important parameter for zinc tolerance and PDF1 tissue specificity could be an important factor to consider. Moreover, post-transcriptional and post-translational regulation might occur. Studies on these modifications are now the further questions in order to understand the contribution of the different PDF1s to zinc tolerance

Les légumineuses en milieu carencé en azote, établissent une relation symbiotique avec les bactéries du sol appelées rhizobia. Cette interaction conduit à la formation d’un nouvel organe racinaire, la nodosité. Au sein de celle-ci, les rhizobia se différencient en bactéroïdes fixant l’azote atmosphérique au profit de la plante. La colonisation massive et chronique des cellules symbiotiques de nodosités par les rhizobia ne déclenche aucune réaction de défense visible. Au laboratoire nous avons isolé deux mutants symbiotiques développant des réactions de défense dans les nodosités de Medicago truncatula indiquant qu’il existe un contrôle strict de l’immunité dans cet organe. L’objectif de cette thèse est de comprendre comment l’immunité symbiotique contrôle les voies de signalisation hormonales de défense afin d’héberger le partenaire symbiotique et de trouver de nouveaux outils pour mieux comprendre les mécanismes de défense. Pour cela, des approches moléculaires, pharmacologiques et génétiques sont utilisées. Les résultats obtenus dans cette thèse suggèrent que les mécanismes de défense adoptés par M. truncatula varient en fonction de l’écotype de la plante. L’écotype A17 exploite deux voies de résistance : la voie de la sénescence et la voie des réactions de défense. Cependant, l’écotype R108 n’exploite que la voie des réactions de défense. Ce travail suggère également que chez M. truncatula A17, la protéine SymCRK réprime la voie de signalisation de l’acide jasmonique qui conduit au déclenchement de la sénescence, tandis que la protéine DNF2 réprime principalement la voie de signalisation de l’acide salicylique qui conduit au déclenchement des réactions de défense et aussi réprime secondairement la voie acide jasmonique. Chez M. truncatula R108, les deux protéines SymCRK et DNF2 contrôlent les voies de signalisation de l’acide salicylique et de l’éthylène, avec DNF2 contrôlant préférentiellement la voie acide salicylique et SymCRK contrôlant préférentiellement la voie éthylène. Cette thèse suggère aussi l’implication des protéines R dans le contrôle de l’accommodation intracellulaire des rhizobia. Un gène CNL-5 semble être impliqué dans le contrôle de l’infection des cellules symbiotiques, et deux autres gènes CNL-4 et TNL-2 semblent être impliqués dans le contrôle de l’efficacité de l’infection. Finalement, cette thèse a permis d’isoler une souche bactérienne E. adhaerens, capable se comporter comme un symbiote ou comme un pathogène pour plusieurs espèces de Medicago
When grown under limited nitrogen resources, legumes establish a symbiotic relationship with soil bacteria called rhizobia. This interaction leads to the formation of a new root organ, the nodules. Within nodules, rhizobia differentiate into bacteroids and fix atmospheric nitrogen for the plant. The massive and chronic colonization of nodule symbiotic cells by the rhizobia does not trigger any visible defense reactions. In the laboratory, we previously isolated two symbiotic mutants developing defense reactions in Medicago truncatula nodules, indicating a strict control of the nodule immunity. The thesis project aimed first to understand the relationship between symbiotic genes and immunity mediated by defense hormones and second to find new tools to help to better understand nodules defense mechanisms. For this, molecular, pharmacological and genetic approaches were used. The results obtained in this thesis suggest that the defense mechanisms adopted by M. truncatula vary depending on the plant ecotypes. The A17 ecotype uses two resistance pathways; senescence and defense reactions. While, the R108 ecotype uses defense reactions pathway to fight against rhizobia. This work also suggests that in M. truncatula A17, the SymCRK protein represses jasmonic acid signalling. This work also suggests that in M. truncatula A17, the SymCRK protein represses the jasmonic acid signaling pathway involved in senescence triggering. In contrast, the DNF2 protein mainly represses the salicylic acid signaling pathway involved in defense reactions and also represses the JA pathway. In M. truncatula R108, the two proteins SymCRK and DNF2 control salicylic acid and ethylene signaling pathways involved in triggering defense reactions. DNF2 controls the salicylic acid pathway more than the ethylene signaling pathway, while, SymCRK controls more the ethylene pathway than the salicylic acid signaling pathway in nodules. This thesis also suggests the involvement of R proteins in the control of intracellular accommodation of rhizobia. The intracellular receptor CNL-5 appears to be involved in controlling the infection of symbiotic cells and the receptors CNL-4 and TNL-2 genes appear to be involved in controlling the infection efficacy of the symbiotic cells. Finally, this thesis allowed the identification of a new bacterial strain E. adhaerens, capable of behaving as a symbiont or as a pathogen for several Medicago species. This can be used as a tool to study the nodules defense induction and to understand the rhizobia intracellular accommodation in symbiotic cells

D.Phil. (Biochemistry)
This thesis concerns a study of the effect of isonitrosoacetophenone on plant metabolism. Three different systems were investigated; cultured tobacco and sorghum cells as well as Arabidopsis thaliana plants, and a metabolomic approach was followed. Unlike most scientific studies, metabolomics is a discipline which is not driven by a specific hypothesis, but rather by the obtained data to add scientific insights to the topic under investigation. As such, the current study lacks a definite overarching hypothesis, but specific objectives were outlined and answered in each experimental chapter. This thesis is therefore presented as a compilation of nine chapters in which experimental/research work is described in Chapter 3- 8. It is important to note that each chapter is presented in accordance with the guidelines for the respective journal in which the corresponding manuscript was published or submitted to.

M.Sc. (Biochemistry)
During microbial invasion, a variety of defense responses are induced in host plants. In order for host plants to combat potential diseases induced by microbes, they must be equipped with pattern recognition receptors (PRRs) localized at the cell surface, since such receptors enable the perception of conserved microbial epitopes termed microbe/pathogen-associated molecular patterns (M/PAMPs), thereby resulting in the activation of plant innate immunity via M/PAMP-triggered immunity (P/MTI). Lipopolysaccharide (LPS) is the major component of the outer leaflet of the external membrane of Gram-negative bacteria. This thermo-stable lipoglycan is exposed towards the external environment and plays an important role in bacterial adaptation to external surroundings. LPS is recognized as a major M/PAMP in plants, and thus potentiates or elicits defense-related responses such as the production of antimicrobial compounds and the expression of immune response genes. One of the most widely investigated effects of LPS on plants is its ability to prevent and/or suppress the hypersensitive response (HR) induced by an array of bacteria. The HR is a programmed cell death response which ends in a local necrosis of plant tissue, thereby resulting in a reduced number of viable bacteria that can further promote disease progression in the host.