Phytochromes (PHYs) and plant hormones have been emerging as important regulators of fleshy fruit physiology and quality traits; however, the relevance of PHY-hormonal signaling crosstalk in controlling fruit development and metabolism remains elusive. This Thesis assesses the role of PHYs and their interplay with auxins, cytokinins and ethylene during the regulation of tomato (Solanum lycopersicum) fruit development and ripening, with a focus on the control of the plastid biogenesis, sugar metabolism and carotenoid accumulation. In Chapter I, we present evidence that the deficiency in PHY chromophore phytochromobilin (PΦB) biosynthesis, which leads to a global deficiency in functional PHYs, represses fruit chloroplast biogenesis in immature fruits and inhibits fruit sugar accumulation by transcriptionally downregulating sink- and starch biosynthesis-related enzymes. Genetic and physiological evidence suggested the involvement of both auxins and cytokinins as mediators of the negative impact of PΦB deficiency on fruit sink strength and chloroplast formation. During the ripening phase, PΦB deficiency was shown to delay the rise in climacteric ethylene production, affecting the ripening initiation rather than its progression. PHY-hormonal signaling crosstalk was shown to be active not only in the more externally positioned fruit tissues (i.e., pericarp) but also in the most inner fruit regions (i.e., columella). We, therefore, concluded that the global deficiency in functional PHY drastically affects fruit sugar metabolism, chloroplast formation as well as the timing of ripening via an intricate interplay involving phytochromes, auxins, cytokinins and ethylene. In Chapter II, we employed fruit-specific RNAi-mediated silencing of PHY genes to shed light on the specific role played by fruit-localized PHYs and their downstream signaling cascades on tomato fruit physiology and quality traits. Data revealed that fruit-localized SlPHYB2 negatively regulates chlorophyll accumulation in immature fruits whereas SlPHYA positively influences the plastid division machinery. Both SlPHYA and SlPHYB2 were shown to play overlapping, yet distinct, roles in controlling fruit starch metabolism and carotenoid biosynthesis. Our data implicated cytokinin signaling-related proteins as mediators of the SlPHYA-dependent regulation of plastid division machinery, and specific AUXIN RESPONSE FACTORs as intermediates in the PHY-mediated regulation of fruit sugar and carotenoid metabolisms. We concluded that fruit-localized SlPHYA- and SlPHYB2-mediated light perception regulate fruit plastid biogenesis as well as sugar and carotenoid metabolisms via coordinated changes in key components of both auxin and cytokinin signaling cascades. Altogether, this study brings important insights into the combined action of PHYs and hormones in the control of fruit plastid biogenesis and highlights that the interplay between PHY-hormonal signaling cascades influences essential features of tomato fruit quality, such as the sugar and carotenoid accumulation / Fitocromos (PHYs) e fitormônios têm sido caracterizados como importantes reguladores da fisiologia e qualidade de frutos carnosos; todavia, a importância de interações entre a sinalização hormonal e dos PHYs no controle do desenvolvimento e metabolismo de frutos ainda permanece pouco elucidada. Este trabalho de Tese avaliou o papel dos PHYs e das suas interações com as auxinas, as citocininas e o etileno sobre a regulação do desenvolvimento e amadurecimento de frutos de tomateiro (Solanum lycopersicum), particularmente no que tange ao controle da biogênese plastidial e metabolismos de açúcares e de carotenoides. No Capítulo I são apresentadas evidências de que a deficiência na produção de fitocromobilina (PΦB), a qual resulta numa deficiência global in PHYs funcionais, impacta negativamente a biogênese de cloroplastos em frutos imaturos e inibe o acúmulo de açúcares por meio da repressão transcricional de enzimas relacionadas a biossíntese de amido e força de dreno nos frutos. Evidências genéticas e fisiológicas indicaram o envolvimento tanto das auxinas quanto das citocininas como mediadoras do impacto negativo da deficiência de PΦB sobre a força de dreno dos frutos bem como na formação de cloroplastos. Durante a fase de amadurecimento, a deficiência em PΦB atrasou a produção climatérica de etileno, afetando o início do amadurecimento mas não a sua progressão. As interações entre PHYs e hormônios mostraram-se ativas não apenas nos tecidos posicionados mais externamente (i.e., pericarpo) mas também nas regiões mais internas do fruto (i.e., columela). Conclui-se, portanto, que a deficiência global em PHYs funcionais afeta drasticamente o metabolismo de açucares, formação de cloroplastos, bem como o tempo de amadurecimento através de uma interação complexa envolvendo fitocromos, auxinas, citocininas e etileno. No Capítulo II utilizamos o silenciamento fruto-específico de PHYs a fim de desvendar de que forma a fisiologia e parâmetros de qualidade do tomate seriam regulados por PHYs presentes no próprio fruto. Os dados obtidos revelaram que moléculas de SlPHYB2 presentes no próprio fruto regulam negativamente o acúmulo de clorofilas nos frutos imaturos, já as de SlPHYA influenciam positivamente a maquinaria de divisão plastidial, e tanto SlPHYA quanto SlPHYB2 desempenham papel sobrepostos, porém distintos, no controle do metabolismo de amido e acúmulo de carotenoides em frutos de tomateiro. Evidências sugerem que proteínas relacionadas à sinalização de citocininas atuariam como mediadoras do impacto de SlPHYA sobre a maquinaria de divisão plastidial, e que AUXIN RESPONSE FACTORs específicos seriam intermediários no controle dos PHYs sobre os metabolismos de açúcares e carotenoides. Conclui-se, dessa forma, que a percepção de luz mediada por moléculas de SlPHYA e SlPHYB2 presentes no próprio fruto regulam a biogênese plastidial e os metabolismos de açúcares e carotenoides por meio de alterações coordenadas em componentes chaves das cascatas de sinalização de auxinas e citocininas. Quando combinados, os dados obtidos neste estudo apresentam novidades importantes sobre a ação conjunta de PHYs e fitormônios no controle da biogênese plastidial e demonstram que a interação entre esses sinalizadores influencia características essenciais da qualidade de frutos de tomateiro, tais como o acúmulo de açúcares e de carotenoides

Chloroplasts are organelles that convert light energy to chemical energy through photosynthesis. The movement of chloroplasts within the cell for the optimization of light absorption is crucial for plant survival. Cellular motor proteins and cytoskeletal tracks can facilitate transport of organelles. As an ancient superfamily of microtubule-dependent motors, kinesins participate in various cellular activities including cytokinesis, vesicle and organelle movements. Based on phylogenetic relationships and functional analysis, the kinesin superfamily has been subdivided into more than 14 families, most of which can be found in plants. With the ever increasing amount of genomic information, it is important and beneficial to systematically characterize and document kinesins within an organism. As a result of my collaborative work with other members of the Vidali lab, a detailed phylogenetic characterization of the 76 kinesins of the kinesin superfamily in the moss Physcomitrella patens is reported here. We found a remarkable conservation of families and subfamily classes with Arabidopsis, which is important for future comparative analyses of functions. Some of the families are composed of fewer members, while other families are greatly expanded in moss. To improve the comparison between species, and to simplify communication between research groups, we proposed a classification of subfamilies based on our phylogenetic analysis. As part of my efforts in studying chloroplasts motility, I investigated the function of two members of Physcomitrella kinesin family 14 class V proteins, Ppkin14-Va and -Vb. These two proteins are orthologs of the Arabidopsis KAC proteins which mediate actin-based chloroplast movement in Arabidopsis thaliana. In contrast, in the Physcomitrella both actin filaments (AFs) and microtubules (MTs) participate in chloroplast movement. Our results show that Ppkin14-Vs are important for maintaining chloroplast dispersion. They also function during chloroplast light avoidance responses via an AF-dependent, rather than MT-dependent mechanism. Although two Ppkin14- Vs do not act as MT-based motors, our phylogenetic study on moss kinesins provides an important source of information to track other potential kinesins that are predicted to move chloroplasts on MTs.

Biotechnology of microalgae is a fast-growing field and several species have become targets for transgenic manipulation. Microalgae provide low-cost and scalable production platforms for manufacturing recombinant proteins and other high value products. However, the exploitation of microalgae as expression systems is restricted by the low yield of recombinant proteins and the limited availability of tools for the genetic manipulation of commercially important species. This thesis explores transgene instability and gene autoregulation as causes for low recombinant protein accumulation in the chloroplast of Chlamydomonas reinhardtii and describes the isolation of a mutant phytoene desaturase (PDS) gene which confers resistance to the herbicide norflurazon for future use as a selection marker for the marine microalga Dunaliella tertiolecta. Recombination between short dispersed DNA repeats (SDR) found in the chloroplast genome of C. reinhardtii was identified as a cause of transgene instability. The genes coding for β-glucuronidase (GUS) and peridinin-chlorophyll binding protein (PCP) were inserted in the chloroplast genome next to the atpB 3' UTR by homologous recombination. Recombination of a 30bp SDR located within the 3' UTR of atpB was identified as the cause of transgene excision in the transplastomic lines. Such transgene instability was tackled by replacing the 3' UTR of atpB with the rbcL 3' UTR from D. tertiolecta. Using this 3'UTR sequence from a different species produced a photosynthetic strain and prevented excision of the transgene by SDR recombination in all transfomants. Very low levels of recombinant GUS and PCP accumulated in chloroplast transformants when using the psbD 5' regulatory region to drive their expression. To address low levels of accumulation caused by regulatory pathways that inhibit transgene expression, I have engineered the chloroplast genome of a non-photosynthetic atpB mutant of C. reinhardtii by replacing the endogenous psbD promoter and 5'UTR with the promoter and 5'UTR of psbA. The engineered strain was subsequently transformed with the wildtype atpB and two different reporter genes driven by the psbD regulatory regions: gusA and kat, which code for GUS and the fluorescent protein Katushka respectively. Analysis of the transformants showed that accumulation of recombinant proteins in our engineered strain was 10 to 20 fold higher than in the nonengineered cells. Most of the selectable markers used in plants and algae are inefficient in Dunaliella, which is naturally resistant to many of the antibiotics used for the selection of transformants. Norflurazon inhibits PDS, an essential enzyme for carotenoid biosynthesis. Using forward genetics I have isolated, sequenced and characterised mutant PDS alleles conferring norflurazon resistance in D. tertiolecta. Independent mutations in pds, leading to substitutions R265C, S472L, S472F and L502F, all result in high resistance to norflurazon but differ in sensitivity to other bleaching herbicides. By mapping the four amino acid substitutions on 3D models of D. tertiolecta PDS I determined that R265C, S472L, S472F and L502F, cluster together in proximity to a Rossman-like domain and to aminoacids F128 and V469, previously reported to confer norflurazon resistance. This suggests that the mode of action of norflurazon is by competition with flavin adenine dinucleotide (FAD) for its binding site. A unique aspect of the R265C substitution is its negative cross-resistance to diflufenican and beflutamid which could be advantageous for its use as a positive/negative selection marker for transformation.

Plastids are organelles responsible for several essential aspects for plant development, like photosynthesis, nitrogen assimilation and synthesis of several compounds of secondary metabolism. Chloroplasts differentiation and activity are highly regulated by light, and several proteins and mechanisms involved in these processes have been characterized. The GOLDEN 2-LIKE (GLK) transcription factors controls the expression of several genes related to photosynthesis, plastid biogenesis and maintenance. Solanum lycopersicum genome harbors two copies of this gene, SlGLK1 and SlGLK2 and, although they are functionally redundant, their expression pattern is different, once SlGLK1 predominates in leaves, while only SlGLK2 is expressed in fruit, precisely at the pedicel region. During tomato domestication, selection for varieties that ripened evenly resulted in the fixation of uniform ripening mutation (Slglk2) in most cultivated varieties, resulting in alterations in fruit metabolic composition. In this context, the objective of this work was to functionally characterize SlGLK2 gene aiming to understand in which way phytochrome mediated light and phytohormones, particularly auxins and cytokinins, regulates this gene expression, and how SlGLK2 presence affects fruit nutritional quality. To achieve this, a detailed transcriptional profile of SlGLK2 was performed in fruits of wild plants, Slglk2 mutant and plants deficient for light perception or hormonal signaling. The effect of SlGLK2 over nutritional quality was evaluated by characterizing carbon and vitamin E metabolism. Additionally, reporter protein GUS activity was quantified in transgenic plants that express uidA gene under control of promoters responsive to cytokinins or auxins in SlGLK2 or Slglk2 genotypes, to analyze if hormonal activity is affected by SlGLK2 presence. Finally, in order to verify if the presence of SlGLK2 is sufficient to reverse the chlorotic phenotype of the mutant aurea, promoting the differentiation and plastidial maturation even in the absence of functional phytochromes, transgenic lines were generated by overexpressing the SlGLK2 gene on aurea-Slglk2 genetic background. The integrated data analysis allowed us to conclude that the content of soluble sugars and vitamin E correlate with the expression of SlGLK2, that the expression of SlGLK2 is repressed by auxins, that SlGLK2 positively participates in the signaling of cytokinins, and that its overexpression partially reverts the phenotype of the aurea-Slglk2 mutant fruits. The results obtained in this work contributes to a better understanding of the regulatory network that interconnects SlGLK2 gene, phytohormones and light, promoting the plastidial activity and consequently, determining the nutritional quality of the tomato fruit, an important component of the human diet / Os plastídios são organelas responsáveis por diversos aspectos essenciais do desenvolvimento das plantas como a fotossíntese, assimilação de nitrogênio e síntese de diversos compostos do metabolismo secundário. A diferenciação e atividade dos cloroplastos são altamente reguladas pela luz, e diversas proteínas e mecanismos envolvidos nestes processos têm sido caracterizados. Os fatores de transcrição GOLDEN 2-LIKE (GLKs) controlam a expressão de diversos genes relacionados à fotossíntese, biogênese e manutenção plastidial. Solanum lycopersicum possui duas cópias desses genes, SlGLK1 e SlGLK2 e, embora sejam funcionalmente redundantes, seu padrão de expressão é diferente, uma vez que SlGLK1 predomina nas folhas ao passo que SlGLK2 é expresso apenas nos frutos, mais precisamente na região pedicelar. Durante o processo de domesticação do tomateiro, a seleção de variedades de amadurecimento uniforme resultou na fixação da mutação uniform ripening (Slglk2) na maioria das variedades cultivadas, resultando em mudanças na composição metabólica dos frutos. Neste contexto, este trabalho teve como objetivo geral caracterizar funcionalmente o gene SlGLK2 visando compreender de que forma a luz (mediada por fitocromos) e os fitormônios (particularmente citocininas e auxinas), regulam a expressão deste gene e como a presença de SlGLK2 afeta a qualidade nutricional dos frutos. Para isso, foi realizado um detalhado perfil transcricional de SlGLK2 em frutos de plantas selvagens, Slglk2 mutantes e deficientes para a percepção luminosa e para a sinalização hormonal. O efeito de SlGLK2 sobre a qualidade nutricional foi avaliado caracterizando o metabolismo de carbono e de vitamina E. Adicionalmente, foi quantificada a atividade da proteína repórter GUS em plantas transgênicas que expressam o gene uidA sob controle de promotores responsivos a citocininas ou auxinas em plantas com genótipo SlGLK2 ou Slglk2 para analisar se a atividade hormonal é afetada pela presença de SlGLK2. Finalmente, com o intuito de verificar se a presença de SlGLK2 é suficiente para reverter o fenótipo clorótico do mutante aurea, promovendo a diferenciação e maturação plastidial mesmo na ausência de fitocromos funcionais, foram geradas linhagens transgênicas sobreexpressando o gene SlGLK2 em fundo genético aurea-Slglk2. A Análise dos resultados permitiu concluir que o conteúdo de açúcares solúveis e vitamina E correlacionam com a expressão de SlGLK2, que a expressão de SlGLK2 é reprimida por auxinas, que SlGLK2 participa positivamente da sinalização de citocininas, e que a sua sobreexpressão reverte, parcialmente, o fenótipo dos frutos da mutante aurea-Slglk2. Os resultados obtidos nos levam a uma melhor compreensão da rede regulatória que interconecta o gene SlGLK2, os fitormônios e a luz promovendo a atividade plastidial e, por consequência, determinando a qualidade nutricional dos frutos de tomateiro, importante componente da dieta humana

The fern genus Pteridium comprises a number of closely related species distributed throughout the world. Collectively they are called bracken ferns and have historically been treated as a single species, Pteridium aquilinum. Bracken is notorious as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily cultured and has become one of the most intensively studied ferns. Bracken has been used as a model system for the study of the fern life cycle, fern gametophyte development, the pheromonal mechanism of sex determination, toxicology, invasion ecology, and climate change. This dissertation places bracken within a global evolutionary perspective and establishes bracken as an emerging system for evolutionary genomics in ferns. Bracken samples from around the world were examined for chloroplast DNA variation to infer historical phylogenetic and biogeographic evolutionary events. New high-throughput DNA sequencing technologies and bioinformatic approaches were used to determine the complete chloroplast genome sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to identify gene sequences that are expressed in the gametophyte stage of the fern life cycle. These data represent an important genomic resource in ferns and were examined within a functional and evolutionary perspective. Several novel approaches and analyses were developed in the course of this research. Results presented in this dissertation provide novel insights into fern biology and land plant evolution.

Tracking seed dispersal using traditional, direct measurement approaches is difficult and generally underestimates dispersal distances. Variation in chloroplast haplotypes (cpDNA) offers a way to trace past seed dispersal and to make inferences about factors contributing to present patterns of dispersal. Although cpDNA generally has low levels of intraspecific variation, this can be overcome by assaying the whole chloroplast genome. Whole-genome sequencing is more expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among SNPs are lost in pooled samples and treating SNP frequencies as independent estimates of variation provides biased estimates of genetic distance. I have developed an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP frequencies from pooled samples based on network topology, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially-constructed test networks and pools, and pooled samples of Lasthenia californica (California goldfields) from Whetstone Prairie, in Southern Oregon, USA. In test networks, CallHap reliably recovered network topologies and haplotype frequencies. Overall, the CallHap pipeline allows for the efficient use of resources for estimation of genetic distance for studies using non-recombining, whole-genome haplotypes, such as intra-specific variation in chloroplast, mitochondrial, bacterial, or viral DNA.

Extensive research into enzyme-induced bio-conversion of lignocellulose to soluble sugars has been conducted and research continues in this area. Several approaches have been taken to attempt to alleviate the economic problems associated with utilisation of lignocellulose in fuel ethanol production. By expressing cellulase genes in planta, it is hoped that the cost of enzyme-mediated hydrolysis of cellulose to its soluble sugar monomers, will be reduced. Some accomplishments have been made in this area using nuclear genetic transformation (Abdeev et al., 2003; Abdeev et al., 2004; Austin-Phillips et al., 1999; Biswas et al., 2006; Dai et al., 2000a,b; Dai et al., 2005; Jin et al., 2003; Kawazu et al., 1999; Sakka et al., 2000; Ziegelhoffer et al., 1999; Ziegelhoffer et al., 2001; Ziegler et al., 2000), but more research is required to bring the levels of cellulase enzyme expression in plants to levels that will make the process economically competitive. Chloroplasts of N. tabacum were selected as a target for transformation for high level expression due to their extremely high rates of transcription and translation. These were transformed with two genes, the e1 gene from A. cellulolyticus, and the cbh1 gene from T. reesei. Further aims included the investigation of the effects of using different promoters, and the novel use of both nuclear and chloroplast-based expression in a single plant, on the level of protein production in the heterologous host. Heterologous expression of the cbh1 gene was not successful. This is thought to be due to toxicity of the protein in a prokaryotic environment. Future studies should focus on trying to avoid this toxicity by targeting of the chloroplast-expressed enzyme to specific tissues, such as the thylakoid membrane, for containment, creating a codon-optimised synthetic gene that better mimics the codon usage of the plant to be used for expression, or placing the expression under a reactive cascade that is only activated upon exposure to an external trigger. Heterologous expression of the full length gene for E1 from A. cellulolyticus was successful. Chloroplast homology vectors under the constitutive promoter Prrn, and the inducible promoter T7, were constructed and these were used to successfully transform N. tabacum cv. Petit Havana chloroplasts. Stable transgenic plants were produced and evaluated by a variety of means, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 3122 ± 466 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 0.35% ± 0.06 of the total soluble protein. Lastly, chloroplast transformation was combined with nuclear transformation to create novel dual-transgenic plants simultaneously expressing E1 from both the nuclear and chloroplast genomes. The combination of these technologies was very successful, with the heterologously expressed enzyme showing activity against the soluble substrate analogue MUC of up to 35706 ± 955 pmol 4-MU/mg TSP/min and an E1 accumulation level of up to 4.78% ± 0.13 of the total soluble protein, and provides a new approach for increasing the accumulation levels of plant-produced cellulase enzymes.

<p>This investigation addresses how photosynthetic efficiency is affected when pea (Pisum sativum) plants are restricted to a sole nitrogen source (i.e. ammonium or nitrate). The pea plants were watered with different nutrient solutions without NO3- or NH4+ for different time-periods in order to assay for nitrogen content. The soluble ammonium and nitrate content was measured throughout the entire growth period. No major differences were observed in nitrogen content during the starvation period up to 25 days. For technical reasons, cultivation of plants could not be extended beyond this time. The chloroplasts and thylakoids were isolated after 25 days and assayed for chlorophyll contents and photosynthetic activity.</p><p>The outcome of these tests indicates a small but unambiguous decrease in the photosynthesis activity for all treatments, relative the control.</p>

<p>The great difference in the level of chloroplast variation and its geographic structure among the three main species studied here demonstrates that forest species do not form a homogeneous group. Hazel shows a genetic structure similar to many other thermophilous species and this structure, in combination with fossil evidence, indicates that the post-glacial colonisation of most of Europe originated in a refugium in western France while the Balkan and Italy were colonised from a south-eastern refugium.</p><p>In sallow and silver birch the chloroplast DNA variation and its structure does not fit with a scenario of glacial restriction to southern refugia and survival at intermediate latitudes is suggested for both species. The chloroplast DNA variation in silver birch suggests the presence of one western and one eastern European post-glacial colonisation route and limited contribution of southern populations in the colonisation of the rest of Europe. Unique haplotypes by the Ural Mountains indicates the possibility of a separate glacial origin of these populations.</p><p>The study of chloroplast DNA in species closely related to sallow and silver birch indicate that extensive hybridisation and cytoplasmic gene flow occurs within both the Salix and Betula genera in Europe. The nuclear and chloroplast phylogenies of 14 Betula species were not in complete agreement with each other or with the classical division of the Betula genus into subgenera or sections. The phylogenetic structure implies that hybridisation has played a role in the evolution of the Betula genus.</p><p>This thesis focuses on the chloroplast DNA variation in three forest tree genera: Corylus, Betula and Salix. Chloroplast PCR-RFLP is used to evaluate the post-glacial history of hazel, Corylus avellana, silver birch, Betula pendula and sallow, Salix caprea and to explore the possibility of introgression in the Salix and Betula genera. In addition, the chloroplast matK gene, its flanking regions and the nuclear ADH gene were used to study the phylogenetic relationships within the Betula genus.</p>

<p>The genus <i>Arnica</i> (Asteraceae) was investigated for phylogenetic relationships and sesquiterpene lactone (STL) content with the aims to trace the evolutionary history of the genus and to investigate possible congruencies between DNA sequence data, secondary chemistry, and biological activity. </p><p>Complex evolutionary patterns in <i>Arnica</i> are evident from phylogenetic analyses of chloroplast regions (the <i>rpl16</i> and <i>rps16</i> introns and the <i>psbA–trnH</i>, <i>ycf4–cemA</i>, and <i>trnT–L</i> spacers), nuclear ribosomal regions (the internal and external transcribed spacers) and the nuclear low-copy DNA region coding for the second largest subunit of RNA polymerase II (<i>RPB2</i>) between exons 17 and 23. Polymorphism was detected in nuclear ribosomal and low-copy regions<i>,</i> likely caused by polyploidy and agamospermy. Lineage sorting and/or hybridization is a possible explanation for incongruencies between topologies of the different DNA regions. None of the five subgenera in <i>Arnica</i> constitute a monophyletic group according to any of our analyses. </p><p>Sesquiterpene lactone profiles were compared to nuclear ribosomal DNA data using phylogenetic inference and principal component analysis for 33 accessions of 16 species. Clusters supported by both STL chemistry and ribosomal DNA sequence data consist of multiple accessions of the same species (e.g.<i> A montana </i>and<i> A. longifolia</i>), indicating that these species are well defined both genetically and chemically, based on our sampling. Support for subspecies classification of <i>A. chamissonis</i> and <i>A. parryi</i> was found in chemical data. For the first time STLs are reported from subtribe Madiinae, sister to Arniciinae.</p><p>Anti-inflammatory properties, as measured by inhibition of human neutrophil elastase release from neutrophils and inhibition of the binding of transcription factor NF-κB to DNA, were investigated for extracts of 12 <i>Arnica</i> species. <i>Arnica montana</i>, <i>A. chamissonis</i> and <i>A. longifolia</i> accessions show high inhibitory effects in both bioassays. Generally, species with a more diverse STL chemistry also possess the strongest inhibitory activity in the bioassays.</p>