Three general questions are studied regarding plant carbon allocation strategies. (1) The R* Rule states that the superior competitor in a plant community should exclude all others by minimizing available limiting nutrient concentration below the level needed for survival of its competitors. I asked whether a plant carbon allocation strategy that minimizes the concentration of available limiting nutrient is consistent with Lotka's (1922) conjecture that ecosystems should evolve to maximize total energy flow (primary production). (2) In landscapes such as the Everglades, areas of landscape with higher energy flow (primary production) than the surrounding area also have higher available concentrations of limiting nutrient, rather than lower concentrations, which might be expected from the R* rule. I asked whether this pattern can be explained. (3) I asked how optimal allocation of carbon to plant defense allocation strategies might depend on different conditions of nutrient availability, shading, and herbivory. To address all three questions, I used a model revised from the G'DAY model (Comins and McMurtrie 1993) to study tree allocation of carbon resources between foliage, roots, and defense. With regard to the first question, I found that the allocation strategy that leads to minimum concentration of available nutrients is the same as the strategy for which energy flux to roots, rather than total energy flux, is maximized. Further, I found that the strategy that was competitively dominant was neither the strategy for which total energy flux was maximized, nor that for which available nutrient concentration was minimized. With regard to the second question, I found that, if a patch of vegetation on a landscape is able to capture nutrients from the surrounding landscape, for example, through relatively higher evapotranspiration, it could lead to the opposite of what is expected from the R* rule; that is, available limiting nutrient concentration is maximized when carbon flow to the roots is maximized. With regard to the last question, I found that under high herbivory, the optimal plant strategy for allocation of carbon to defense depends on the available nutrient concentration and amount of radiation to the plant, in agreement with some theoretical predictions.

Systemic acquired resistance (SAR) is initiated upon recognition of specific microbial effectors by cognate plant resistance proteins and immunizes distal tissues of plants against secondary infections. SAR involves the generation of a mobile signal at the site of primary infection, which then translocates to and activates defense responses in the distal tissues via some unknown mechanism(s). This study shows that an ACYL CARRIER PROTEIN 4 (ACP4), GLABRA1 (GL1) and ACYL CARRIER BINDING PROTEINS (ACBP) are required for the processing of the mobile SAR signal in distal tissues of Arabidopsis. Although acp4, gl1 and acbp plants generate the mobile signal, they are unable to respond to this signal to induce systemic immunity. A defective SAR in acp4, gl1 and acbp plants is not associated with salicylic acid (SA)-, methyl SA-, or jasmonic acid-mediated pathways but is related to the presence of an abnormal cuticle on acp4, gl1 and acbp plants. Other genetic mutations impairing the cuticle also compromised SAR. An intact cuticle was only necessary during the time when the mobile signal is generated and translocated to the distal tissues. A novel role for the plant cuticle as the site for SAR-related molecular signaling is demonstrated.

VIANA, Carolina de Araújo. Caracterização bioquímica e atividades biológicas de quitinases laticíferas de Calotropis procera. 2015. 181 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Vitor Campos (vitband@gmail.com) on 2016-09-01T23:13:32Z No. of bitstreams: 1 2015_tese_caviana.pdf: 4184651 bytes, checksum: cb48948d5a8582e15dd64207712d9789 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-02T21:14:29Z (GMT) No. of bitstreams: 1 2015_tese_caviana.pdf: 4184651 bytes, checksum: cb48948d5a8582e15dd64207712d9789 (MD5) / Made available in DSpace on 2016-09-02T21:14:29Z (GMT). No. of bitstreams: 1 2015_tese_caviana.pdf: 4184651 bytes, checksum: cb48948d5a8582e15dd64207712d9789 (MD5) Previous issue date: 2015 / Calotropis procera is a latificer plant in the Apocynaceae family. Defensive proteins with activity against phytophagous insects and phytopathogenic fungi in latex have been described. Latex’s proteic fraction also performs pharmacological activities, such as selective cytotoxic effect in carcinogenic cells. From the proteic fraction effective against neoplastic cells, this work had the goal to identify and further characterize cytotoxic protein(s) biochemically, biologically, and evaluate their biotechnological prospects. Soluble proteins in latex (LP) have been fractioned in ion-exchange matrix, and the fraction capable of cytotoxicity was further fractioned in another ion-exchange matrix (Mono-Q) coupled to a medium-pressure system. P4 was identified as cytotoxic fraction, showing an IC50 of 2.2, 1.2 and 2.9 mg/mL for the cell lines HCT-116, Ovcar-8 and SF-295, respectively. Proteomic analysis of the cytotoxic fraction by two-dimensional electrophoresis allowed 15 spots to be identified, comprising acid proteins (pI among 4 and 6) with 30 kDa apparent molecular weight. All spots were identified as chitinases when evaluated by mass spectrometry, and showed intact mass of 27.4 kDa. The sample reacted positively to the Schiff reagent, suggesting glycosilations. The carbohydrate content was estimated at 12.8%. The amino-terminal sequence obtained (1QPVMNLEYPRYLNDINDYRDDNNYD28) revealed 50% similarity with Solanum lycopersicum, Oryza sativa and Nicotiana tomentosiformes chitinases, among others. The enzymes showed strong chitinolytic activity, with optimal pH varying between 5-6 and optimal temperature of 25 °C. The chitinolytic activity was diminished when treated with increasing concentrations of DTT (3, 10 and 30 mM), which suggests the presence of disulfide bonds stabilizing the enzyme. Circular dichroism analyses indicate a larger presence of alpha helices in the structure and that the isoforms has low resistance to pH and temperature variation. High-resolution images generated through atomic force microscopy suggested sample homogeneity and a hexameric configuration. The chitinases did not inhibit mycelial growth of phytopathogenic fungi Fusarium oxysporum and Colletotrichum gloeosporioides, however reduced the germination C. gloeosporioides spores. In consonancy the chitinases did not induce spore oxidative stress, but caused a slight change in membrane permeability of the evaluated spores. Laticifer chitinases (0.1% w/w) showed a strong deleterious effect on the phytophagous insect Callosobruchus maculatus. Effects produced a 57% survival reduction, larval weight reduction (7.8 mg ± 0.2 / 4.0 ± 0.8), adult insets emergence reduction (50%), in addition to prolonging the mean maturation time from 28 to 33 days. The chitinases (2 mg/Kg, i.v.) showed a strong anti-inflammatory activity, reducing 95% of neutrophil infiltration into the peritoneal cavity in mouse in an inflammation induced by carrageenan assay. L-NAME and Aminoguanidine, two inhibitors of nitric oxide synthase, reversed this effect, possibly indicating involvement of nitric oxide in the effects observed. This action was associated to a reduction of pro-inflammatory cytokines TNF-α and IL-1 levels within the peritoneal cavity and increased serum levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1. The conclusion is reached that many isoforms of chitinases coexist in C. procera latex. These proteins act possibly in defense against insects, though low action against fungi is shown. Laticifer chitinases were identified as latex proteins cytotoxic on neoplastic cells and they have even been able to modulate pro-inflammatory cytokines levels and nitric oxide in an acute inflammation model. C. procera laticifer chitinases represent interesting molecules for biotechnology prospection in plant defense and pharmacology. / Calotropis procera é uma planta laticífera, pertencente à família Apocynaceae. Proteínas de defesa foram descritas no látex com atividade contra insetos fitófagos e fungos fitopatogênicos. A fração proteica do látex também apresenta atividades farmacológicas, dentre tais efeito citotóxico seletivo em células carcinogênicas. A partir da fração proteica efetiva sobre células neoplásicas, o trabalho teve por meta central identificar proteína(s) citotóxica(s) e proceder a sua caracterização bioquímica, biológica e suas perspectivas biotecnológicas. As proteínas solúveis do látex (PL) foram fracionadas em matriz de troca iônica e a fração proteica detentora da citotoxicidade foi refracionada em outra matriz de troca iônica (Mono-Q) acoplada a sistema de média pressão. P4 foi identificado como a fração citotóxica, apresentando uma IC50 de 2,2, 1,2 e 2,9 µg/mL para as linhagens celulares HCT-116, Ovcar-8 e SF-295, respectivamente. A análise proteômica da fração citotóxica por eletroforese bidimensional permitiu identificar 15 spots, contendo proteínas ácidas (pI entre 4 e 6) e com massa molecular aparente de 30 kDa. Quando avaliados por espectrometria de massas todos esses spots foram identificados como quitinases e apresentaram massa intacta de 27,4 kDa. A amostra reagiu positivamente ao reagente de Schiff, sugerindo glicosilações. O teor de carboidratos foi estimado em 12,8%. A sequência amino terminal obtida (1QPVMNLEYPRYLNDINDYRDDNNYD28) revelou 50% de similaridade com quitinases de Solanum lycopersicum, Oryza sativa, Nicotiana tomentosiformes dentre outras. As enzimas apresentaram forte atividade quitinolítica, com pH ótimo variando entre 5-6 e temperatura ótima de 25 °C. A atividade quitinolítica foi reduzida quando tratada com concentrações crescentes de DTT (3, 10 e 30 mM) o que sugere a presença de pontes dissulfeto estabilizadoras da enzima. Análises por dicroísmo circular indicam a presença majoritária de alfa-hélices na estrutura e que as isoformas se mostraram pouco resistentes a variações de temperatura e pH. Imagens de alta resolução geradas por microscopia de força atômica sugeriram homogeneidade na amostra e um arranjo hexamérico foi evidenciado. As quitinases não inibiram o crescimento micelial dos fungos fitopatogênicos Fusarium oxysporum e Colletotrichum gloeosporioides, mas reduziram a germinação de esporos de C. gloeosporioides. Em consonância as quitinases não induziram estresse oxidativo nos esporos, mas causaram leves alterações na permeabilidade membranar dos esporos avaliados. As quitinases laticíferas (0,1 % m/m) apresentaram forte efeito deletério sobre o inseto fitófago Callosobruchus maculatus. Os efeitos produziram 57% de redução na sobrevivência larval, redução do peso de larvas (7,8 mg ± 0,2 /4,0 ± 0,8) e emergência de insetos adultos (50%), além de prolongar de 28 para 33 dias o tempo médio de desenvolvimento de insetos adultos. As quitinases (2 mg/Kg, e.v.) apresentaram forte atividade anti-inflamatória, reduzindo em 95% a infiltração de neutrófilos na cavidade peritoneal em ensaio de inflamação induzido por carragenina em camundongos. Este efeito foi revertido por L-NAME e Aminoguanidina, dois inibidores da enzima óxido nítrico sintase, indicando o possível envolvimento do óxido nítrico no efeito observado. Esta ação foi associada à redução dos níveis das citocinas pró-inflamatórias TNF-α e IL-1 na cavidade peritoneal e do aumento dos níveis séricos das citocinas pró-inflamatórias TNF-α, IL-6 e IL-1. É concluído que várias isoformas de quitinases coexistem no látex de C. procera. Estas proteínas atuam possivelmente na defesa contra insetos, mas têm pouca ação contra fungos. As quitinases laticíferas foram identificadas como as proteínas citotóxicas do látex sobre células neoplásicas e ainda foram capazes de modular os níveis de citocinas pró-inflamatórias e síntese de óxido nítrico em modelo de inflamação aguda. As quitinases do látex de C. procera representam interessantes moléculas para prospecção biotecnológica em defesa vegetal e farmacologia.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Calotropis procera à uma planta laticÃfera, pertencente à famÃlia Apocynaceae. ProteÃnas de defesa foram descritas no lÃtex com atividade contra insetos fitÃfagos e fungos fitopatogÃnicos. A fraÃÃo proteica do lÃtex tambÃm apresenta atividades farmacolÃgicas, dentre tais efeito citotÃxico seletivo em cÃlulas carcinogÃnicas. A partir da fraÃÃo proteica efetiva sobre cÃlulas neoplÃsicas, o trabalho teve por meta central identificar proteÃna(s) citotÃxica(s) e proceder a sua caracterizaÃÃo bioquÃmica, biolÃgica e suas perspectivas biotecnolÃgicas. As proteÃnas solÃveis do lÃtex (PL) foram fracionadas em matriz de troca iÃnica e a fraÃÃo proteica detentora da citotoxicidade foi refracionada em outra matriz de troca iÃnica (Mono-Q) acoplada a sistema de mÃdia pressÃo. P4 foi identificado como a fraÃÃo citotÃxica, apresentando uma IC50 de 2,2, 1,2 e 2,9 Âg/mL para as linhagens celulares HCT-116, Ovcar-8 e SF-295, respectivamente. A anÃlise proteÃmica da fraÃÃo citotÃxica por eletroforese bidimensional permitiu identificar 15 spots, contendo proteÃnas Ãcidas (pI entre 4 e 6) e com massa molecular aparente de 30 kDa. Quando avaliados por espectrometria de massas todos esses spots foram identificados como quitinases e apresentaram massa intacta de 27,4 kDa. A amostra reagiu positivamente ao reagente de Schiff, sugerindo glicosilaÃÃes. O teor de carboidratos foi estimado em 12,8%. A sequÃncia amino terminal obtida (1QPVMNLEYPRYLNDINDYRDDNNYD28) revelou 50% de similaridade com quitinases de Solanum lycopersicum, Oryza sativa, Nicotiana tomentosiformes dentre outras. As enzimas apresentaram forte atividade quitinolÃtica, com pH Ãtimo variando entre 5-6 e temperatura Ãtima de 25 ÂC. A atividade quitinolÃtica foi reduzida quando tratada com concentraÃÃes crescentes de DTT (3, 10 e 30 mM) o que sugere a presenÃa de pontes dissulfeto estabilizadoras da enzima. AnÃlises por dicroÃsmo circular indicam a presenÃa majoritÃria de alfa-hÃlices na estrutura e que as isoformas se mostraram pouco resistentes a variaÃÃes de temperatura e pH. Imagens de alta resoluÃÃo geradas por microscopia de forÃa atÃmica sugeriram homogeneidade na amostra e um arranjo hexamÃrico foi evidenciado. As quitinases nÃo inibiram o crescimento micelial dos fungos fitopatogÃnicos Fusarium oxysporum e Colletotrichum gloeosporioides, mas reduziram a germinaÃÃo de esporos de C. gloeosporioides. Em consonÃncia as quitinases nÃo induziram estresse oxidativo nos esporos, mas causaram leves alteraÃÃes na permeabilidade membranar dos esporos avaliados. As quitinases laticÃferas (0,1 % m/m) apresentaram forte efeito deletÃrio sobre o inseto fitÃfago Callosobruchus maculatus. Os efeitos produziram 57% de reduÃÃo na sobrevivÃncia larval, reduÃÃo do peso de larvas (7,8 mg  0,2 /4,0  0,8) e emergÃncia de insetos adultos (50%), alÃm de prolongar de 28 para 33 dias o tempo mÃdio de desenvolvimento de insetos adultos. As quitinases (2 mg/Kg, e.v.) apresentaram forte atividade anti-inflamatÃria, reduzindo em 95% a infiltraÃÃo de neutrÃfilos na cavidade peritoneal em ensaio de inflamaÃÃo induzido por carragenina em camundongos. Este efeito foi revertido por L-NAME e Aminoguanidina, dois inibidores da enzima Ãxido nÃtrico sintase, indicando o possÃvel envolvimento do Ãxido nÃtrico no efeito observado. Esta aÃÃo foi associada à reduÃÃo dos nÃveis das citocinas prÃ-inflamatÃrias TNF-α e IL-1 na cavidade peritoneal e do aumento dos nÃveis sÃricos das citocinas prÃ-inflamatÃrias TNF-α, IL-6 e IL-1. à concluÃdo que vÃrias isoformas de quitinases coexistem no lÃtex de C. procera. Estas proteÃnas atuam possivelmente na defesa contra insetos, mas tÃm pouca aÃÃo contra fungos. As quitinases laticÃferas foram identificadas como as proteÃnas citotÃxicas do lÃtex sobre cÃlulas neoplÃsicas e ainda foram capazes de modular os nÃveis de citocinas prÃ-inflamatÃrias e sÃntese de Ãxido nÃtrico em modelo de inflamaÃÃo aguda. As quitinases do lÃtex de C. procera representam interessantes molÃculas para prospecÃÃo biotecnolÃgica em defesa vegetal e farmacologia. / Calotropis procera is a latificer plant in the Apocynaceae family. Defensive proteins with activity against phytophagous insects and phytopathogenic fungi in latex have been described. Latexâs proteic fraction also performs pharmacological activities, such as selective cytotoxic effect in carcinogenic cells. From the proteic fraction effective against neoplastic cells, this work had the goal to identify and further characterize cytotoxic protein(s) biochemically, biologically, and evaluate their biotechnological prospects. Soluble proteins in latex (LP) have been fractioned in ion-exchange matrix, and the fraction capable of cytotoxicity was further fractioned in another ion-exchange matrix (Mono-Q) coupled to a medium-pressure system. P4 was identified as cytotoxic fraction, showing an IC50 of 2.2, 1.2 and 2.9 mg/mL for the cell lines HCT-116, Ovcar-8 and SF-295, respectively. Proteomic analysis of the cytotoxic fraction by two-dimensional electrophoresis allowed 15 spots to be identified, comprising acid proteins (pI among 4 and 6) with 30 kDa apparent molecular weight. All spots were identified as chitinases when evaluated by mass spectrometry, and showed intact mass of 27.4 kDa. The sample reacted positively to the Schiff reagent, suggesting glycosilations. The carbohydrate content was estimated at 12.8%. The amino-terminal sequence obtained (1QPVMNLEYPRYLNDINDYRDDNNYD28) revealed 50% similarity with Solanum lycopersicum, Oryza sativa and Nicotiana tomentosiformes chitinases, among others. The enzymes showed strong chitinolytic activity, with optimal pH varying between 5-6 and optimal temperature of 25 ÂC. The chitinolytic activity was diminished when treated with increasing concentrations of DTT (3, 10 and 30 mM), which suggests the presence of disulfide bonds stabilizing the enzyme. Circular dichroism analyses indicate a larger presence of alpha helices in the structure and that the isoforms has low resistance to pH and temperature variation. High-resolution images generated through atomic force microscopy suggested sample homogeneity and a hexameric configuration. The chitinases did not inhibit mycelial growth of phytopathogenic fungi Fusarium oxysporum and Colletotrichum gloeosporioides, however reduced the germination C. gloeosporioides spores. In consonancy the chitinases did not induce spore oxidative stress, but caused a slight change in membrane permeability of the evaluated spores. Laticifer chitinases (0.1% w/w) showed a strong deleterious effect on the phytophagous insect Callosobruchus maculatus. Effects produced a 57% survival reduction, larval weight reduction (7.8 mg  0.2 / 4.0  0.8), adult insets emergence reduction (50%), in addition to prolonging the mean maturation time from 28 to 33 days. The chitinases (2 mg/Kg, i.v.) showed a strong anti-inflammatory activity, reducing 95% of neutrophil infiltration into the peritoneal cavity in mouse in an inflammation induced by carrageenan assay. L-NAME and Aminoguanidine, two inhibitors of nitric oxide synthase, reversed this effect, possibly indicating involvement of nitric oxide in the effects observed. This action was associated to a reduction of pro-inflammatory cytokines TNF-α and IL-1 levels within the peritoneal cavity and increased serum levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1. The conclusion is reached that many isoforms of chitinases coexist in C. procera latex. These proteins act possibly in defense against insects, though low action against fungi is shown. Laticifer chitinases were identified as latex proteins cytotoxic on neoplastic cells and they have even been able to modulate pro-inflammatory cytokines levels and nitric oxide in an acute inflammation model. C. procera laticifer chitinases represent interesting molecules for biotechnology prospection in plant defense and pharmacology.

N-acyl ethanolamines (NAEs) are a class of lipids recently recognized as signaling molecules which are controlled, in part, by their degradation by fatty acid amide hydrolase (FAAH). On the basis of previous studies indicating increased NAE levels in a tobacco cell suspension-xylanase elicitor exposure system and the availability of FAAH mutants, overexpressor and knockout (OE and KO) genotypes in Arabidopsis thaliana, further roles of NAEs in A. thaliana plant defense was investigated. The commonly occurring urban antimicrobial contaminant triclosan (TCS) has been shown to suppress lipid signaling associated with plant defense responses. Thus, a second objective of this study was to determine if TCS exposure specifically interferes with NAE levels. No changes in steady state NAE profiles in A. thaliana-Pseudomonas syringae pv. syringae and A. thaliana-flagellin (bacterial peptide, flg22) challenge systems were seen despite evidence that defense responses were activated in these systems. There was a significant drop in enoyl-ACP reductase (ENR) enzyme activity, which catalyzes the last step in the fatty acid biosynthesis pathway in plants, on exposure of the seedlings to TCS at 10 ppm for 24 h and decreased reactive oxygen species (ROS) production due to flg22 in long term exposure of 0.1 ppm and short term exposure of 5 ppm. However, these responses were not accompanied by significant changes in steady state NAE profiles.

Quaking aspen (Populus tremuloides) is the most widely distributed tree in North America (Lindroth and St Clair 2013), and a keystone species in our western montane forests (Worrall et al. 2015). Aspen has become a model organism for studies of genetics and physiology in woody plants (Bradshaw et al. 2000, Taylor 2002). Aspen is also economicallyimportant (Worrall et al. 2015) – wood is harvested for various uses, its scenic beauty helps sustain the tourism economy in many areas, and it has recently been studied as a possible source of biofuel (Sannigrahi et al. 2010). Aspen is also a species of conservation concern, due to recent large-scale deterioration and decline of many aspen forests in the last two decades (Worrall et al. 2013). Several causal factors have been identified: fire suppression (Calder et al. 2011, Smith et al. 2011), increased ungulate herbivory (Kay and Bartos 2000), disease (Marchetti et al. 2011), and climate change (Worrall et al. 2013). My thesis focuses on two different biotic stressors of aspen: a fungal pathogen and ungulate herbivory. Understanding the relationship between aspen and their biotic stressors adds to our knowledge of aspen ecology and helps manage the increasing risk of decline in our aspen forests. Chapter 1 is a study of the relationship between aspen and a necrotrophic fungal pathogen (Drepanopeziza sp.) during a major disease outbreak in 2015. I quantified the relationship between Drepanopeziza infection severity and aspen leaf functional traits, including morphological, chemical and phenological traits. I found that severe Drepanopeziza infection was associated with low concentrations of a key class of herbivore defense compounds (phenolic glycosides), and strongly associated with early budbreak and leaf-out in aspen stands. The association between infection and early budbreak was likely caused by unusually rainy conditions in May of 2015, which may have exposed leaf tissue to wet conditions that favor thedispersal of Drepanopeziza spores. Chapter 2 is an experiment designed to determine whether the mode and timing of herbivory can influence aspen's defensive response. I specifically asked whether removing leaves, twigs and meristems together and removing leaves alone had unique effects on aspen sucker growth, survival, and phytochemistry. Additionally, I applied these simulated herbivory treatments to suckers on different dates to see whether early- or late-summer herbivory had greater effects on suckers. I found strong mode and timing effects on growth and survival, but not foliar chemistry.

The necrotrophic fungus Botrytis cinerea (B. cinerea) is one of the most aggressive and widespread pathogenic fungi, causing the gray mold disease in over 200 different plants species and severely impacting the quality of harvested fruits. Due to B. cinerea resistance to common synthetic fungicides and their ecological impact, there is a large demand for novel control approaches, such as bio-fungicides. The family of carotenoid-cleavage products, i.e. apocarotenoids, includes important compounds, such as hormones, signaling molecules and growth regulators. Previous studies indicated that the apocarotenoid B-ionone inhibits the growth of some fungal species. In this thesis, we unravel a novel role for B-ionone as a plant regulatory metabolite increasing the resistance against B. cinerea in several plant models. We achieved this by combining phenotypic, transcriptomic, and metabolomic analysis. We show that pretreatment of Arabidopsis plants with B-ionone significantly alleviated the symptoms of B. cinerea infection, modulated hormone homeostasis and affected the interactions between jasmonic acid (JA)/ethylene (ET) and abscisic acid (ABA) hormone signaling pathways β-ionone treatment stimulated JA/ET signaling pathways and repressed the synthesis of ABA upon B. cinerea infection, which reduced the susceptibility of Arabidopsis plants to B. cinerea. To get an overview on the effect of β-ionone on plants at transcript level, we performed an RNA-seq experiment that supported our hypothesis that B-ionone primes and enhances the Arabidopsis immune response to B. cinerea infection and raveled the effect of this volatile on the expression of several transcription factors involved in Arabidopsis immune response to B. cinerea and on transcripts related to cell wall biosynthesis, Map Kinase 3 signaling and hypoxia tolerance. Further experiments performed with transgenic tobacco and tomato plants confirmed the positive role of β-ionone in reducing the symptoms of B. cinerea in green tissues as well as in tomato fruits. The discovery of β-ionone as a new apocarotenoid signaling molecule that regulates plant hormone homeostasis opens up new possibility to control B. cinerea infection and to establish this natural volatile as an environmentally friendly and safe bio-fungicide.

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Sjukdom på grödor orsakad av bakterier kan bidra till ekonomiska förluster för bönder samt brist på mat, därför är det viktigt att utveckla nya hållbara sätt att motverka och behandla grödor mot bakterier. Idag är det mest vanliga tillvägagångssättet antibiotika men detta är inte hållbart p.g.a uppkomst av antibiotikaresistens. Antibiotika är inte heller tillgängligt för alla bönder och grödor då kostnaden blir för hög. Världsbefolkningen växer och om 80 år beräknas det bo mellan 9.9 och 12.7 biljoner (95% konfidens) människor på jorden. Växande befolkning samt ökande klimatförändringar, som torka och höjda temperaturer kräver nya bekämpningsmetoder mot bakterier för att tillgodose behoven i framtiden. Det saknas information om hur växter hanterar och reglerar bakteriella hot, därför är målet med denna studie att bidra med kunskap kring den transkriptionella regleringen av växters immunsystem mot bakterier. För att göra detta har promotorsekvenser hos gener som är förknippade med immunförsvaret i växter undersökts efter konserverade regulatoriska element. En känd receptor, FLS2 har en stor roll i växters försvar mot bakterier och känner igen en peptid från bakteriers flagell. Denna studie har undersökt FLS2 och den sammankopplade receptorn SERK1. Hos FLS2 kunde ingen konserverad modul hittas i uppströmsekvensen, däremot observerades ett 8 bp långt motiv, CAACTTG, i alla undersökta arter. I SERK1 hittades en lång konserverad modul bestående av flera motiv. Både FLS2-motifet och två motiv i SERK1-modulen binds av transkriptionsfaktorn MYC2. För att testa hypotesen att MYC2 bidrar till den transkriptionella regleringen av FLS2 och SERK1 har en experimentell plan utformats, där Nicotiana benthamiana transfekteras av Agrobacterium tumefaciens innehållandes promotorsekvenserna samt generna till transkriptionsfaktorn MYC2. En ökad förståelse kring de olika delarna och mekanismerna som medverkar inom växters immunförsvar kan bidra till den fortsatta forskningen mot hållbara lösningar till att säkra mat i framtiden. / Several factors contribute to the demand of new, sustainable solutions to bring food security to the world population. The United Nations predicts, with a confidence of 95%, that the world population will be between 9.9 and 12.7 billion by year 2100. At the same time plant agriculture as seen today is threatened by climate changes e.g., rising temperatures and more extreme weather conditions. In addition, plant bacterial pathogens reduce yields, and cause losses of over $1 billion dollars worldwide every year to the food production chain. The currently most used and effective treatment against bacterial infections on crops is antibiotics, but this is not a viable alternative for most growers due to increasing antibiotic resistance and the high development, production, and distribution cost. During the upcoming years development of new approaches against bacterial infections on crops is of high importance but currently there are information gaps in the field of plant defense regulation systems. This study was aimed to provide knowledge about the transcriptional regulation of genes that are included in plant immune system towards bacteria. To investigate this, conserved regulatory elements of the upstream sequences of two defense-related plant receptor kinases, FLS2 and SERK1, was searched for in different species. FLS2 is a surface receptor that recognizes a peptide derived from the bacterial flagellin protein, and is part of the pathogen-triggered immunity response of most of higher plants. In FLS2 no conserved module was found but a single motif, CAACTTG, is conserved in all chosen species. In SERK1 a strikingly long and conserved module was found. Both the FLS2 motif and two motifs in the SERK1 module are recognition motifs with MYC2, a transcription factor involved in different plant mechanisms and the regulation of phytohormones like abscisic acid and auxin. To address whether MYC2 is involved in the transcriptional regulation of FLS2, an experimental approach is described, involving transactivation by MYC2 of FLS2 reporter constructs, studies using agroinfiltration in Nicotiana benthamiana. An increased knowledge about the different components and mechanisms of plant defense regulation will help the research towards new bactericides, transgenic plants, and other ways to secure food for upcoming generations.

<p dir="ltr">Plants are capable of perceiving and responding to biotic and abiotic stress. They have evolved a variety of mechanisms to help them recognize and trigger rapid responses to both chemical and mechanical stimuli. These signals coordinate plant growth, development, and innate immune responses. However, we have limited knowledge about how mechanical signals are perceived and transduced during the plant immune response. In this study, we investigated the potential role of PIEZO, a mechanosensitive ion channel that is responsible for cellular mechanotransduction in both the plant and animal kingdoms, during the plant immune responses. Publicly- available RNAseq data revealed that <i>PIEZO</i> expression remained constant and unaltered in response to a variety of phytopathogens or elicitors. We, then, conducted infectious growth assays on <i>piezo</i> mutants in <i>Arabidopsis thaliana</i> plants. Our results indicated that <i>piezo</i> mutants, <i>pzo1-1 </i>and<i> pzo1-5,</i> were more susceptible to <i>Pseudomonas syringae </i>pv. tomato<i> </i>(Pst) DC3000 and to the <i>P. syringae hrcC</i>^<em>-</em> mutant confirming PIEZO’s role in plant defense and PTI. We further explored disease progression with necrotrophic fungi, <i>Alternaria brassisicola</i> and <i>Botrytis cinerea, </i>on <i>piezo</i> mutant plants and found enhanced fungal growth compared to the wild type (Col-0) with <i>Botrytis</i>. Building upon these findings, we probed the role of PIEZO in the growth-defense tradeoff using a root growth inhibition assay with flg22 as the MAMP elicitor. <i>pzo1-1</i> was less sensitive to flg22 treatment with less reduction in root growth compared to wild type whereas <i>pzo1-5</i> shows no difference in reduction compared to Col-0. In addition, we investigated whether PIEZO operates upstream of the main NADPH-oxidase, RBOHD, and the associated oxidative burst that occurs in early defense. There was no significant difference in Reactive Oxygen Species (ROS) production between <i>piezo</i> mutants and the wild type in an apoplastic ROS assay with a MAMP elicitor (flg22) and also with Ca²⁺ flux leaf disk assay. In conclusion, we demonstrated a potential role for PIEZO in plant immune defense responses and the growth-defense tradeoff.</p>