Le silicium est un élément abondant dans la croûte terrestre, et son absorption aurait des effets bénéfiques chez plusieurs plantes, parmi lesquels l'accroissement de la résistance à divers stress biotiques et abiotiques est le mieux documenté. La plus grande partie du silicium absorbé par une plante est polymérisée de façon irréversible sous forme de phytolithes (silice amorphe, SiO2.nH2O). La théorie initialement avancée pour expliquer son rôle, notamment dans le contexte d'interactions plante-agent pathogène, est celle de la « barrière mécanique » qui stipule qu'une couche subcuticulaire de silice empêcherait la pénétration d'agents pathogènes fongiques. Par ailleurs, une certaine quantité demeure en solution dans la plante sous forme d'acide monosilicique (tLjSiC^), et l'on croit maintenant que cette fraction est responsable des propriétés prophylactiques du silicium en modulant les réponses de défense des plantes. L'effet bénéfique du silicium sur l'incidence et l'intensité de maladies fongiques a été décrit dans une variété de pathosystèmes. Toutefois, la nature exacte de l'interaction entre le silicium et les mécanismes de défense ou de résistance est inconnue et demeure un sujet controversé. Nous proposons que le silicium influence l'activité biochimique de composantes responsables de la signalisation intracellulaire post-élicitation, et qu'il a donc un rôle bioactif dans la défense ou la résistance des plantes contre les agents pathogènes fongiques. Afin de mieux comprendre l'effet biologique du silicium et son implication prophylactique, nous avons effectué une analyse transcriptomique complète, à l'aide de la technologie des micropuces d'oligonucléotides, de plants (¥Arabidopsis thaliana sains ou infectés par le blanc (Erysiphe cichoracearum) et traités ou non avec le silicium. L'application de silicium assimilable aux plants sains a significativement altéré l'abondance relative de seulement deux des ca. 40 000 transcrits (28 500 gènes et plus de 10 000 transcrits non annotés) présents sur la micropuce. En revanche, l'inoculation avec Erysiphe cichoracearum s'est traduite par une induction significative de 2 387 gènes et une répression de 1 583 gènes. Pour chaque gène et à l'aide d'un ensemble de procédés informatiques innovateurs, nous avons généré une annotation fonctionnelle unique et simplifiée en utilisant l'annotation finale du génome d'Arabidopsis, l'encyclopédie de Kyoto des gènes et des génomes (KEGG), les sentiers biochimiques « Aracyc », et deux bases de données publiques de facteurs de transcription (DATF et ATTFDB). Les gènes exprimés différentiellement en fonction des traitements ont été classés dans 58 classes fonctionnelles dont 24 classes directement impliquées dans les mécanismes de défense et la pathogenèse. Cette approche a permis d'apprécier dans sa globalité la cascade d'événements transcriptionnels impliqués dans l'interaction Arabidopsis-blanc en présence ou absence de silicium. En réponse à l'infection, 73% des gènes de défense (gènes de résistance, facteurs de transcription liés au stress, gènes impliqués dans la génération de signaux de stress, la synthèse de composés antimicrobiens, le métabolisme de l'oxygène réactif, protéines PR, etc.) ont été significativement induits. En contrepartie, la maladie a affecté certains processus essentiels comme la photosynthèse et le métabolisme énergétique. L'apport de silicium s'est traduit par une atténuation médiane de 25% de la répression des gènes chez les plantes infectées, alors que l'induction attribuable à l'infection a été maintenue à une intensité similaire chez les plantes témoin et celles traitées au silicium. Le rôle du silicium en biologie végétale, son statut essentiel ou bénéfique, son effet direct ou indirect sur la croissance, et son mode d'action bioactif ou mécanique sont discutés depuis plusieurs décennies. Les résultats obtenus dans le cadre de cette étude mènent à conclure que le silicium seul n'a pas d'effet sur le métabolisme de plantes qui croissent dans un environnement contrôlé (sans stress), ce qui supporte l'hypothèse que le silicium n'est pas un élément essentiel. L'apport de silicium assimilable accroît la résistance à l'infection fongique, ce qui se traduit par une atténuation de la répression des gènes impliqués dans le métabolisme énergétique et la photosynthèse; le silicium est donc bénéfique pour une plante en situation de stress, en l'occurrence une infection fongique. Le mode d'action du silicium dans la prophylaxie et dans l'atténuation du stress semble actif car l'induction génique par l'agent pathogène Erysiphe cichoracearum est maintenue chez les plantes traitées; l'influence du silicium ne peut donc pas être réduite à un effet mécanique. / Silicon the second most abundant element in the earth's crust; it would have beneficial effects on the growth of several plants, among which the increase in resistance to various biotic and abiotic stresses is the best documented. Most of the silicon absorbed by a plant is polymerized under the form of phytoliths (amorphous silica, SiO2.nH2O). The initial theory for explaining its role, in particular in the context of plant-pathogen interactions, is that of the « mechanical barrier » which stipulates that a subcuticular layer of silica would prevent the penetration of fungal pathogens. However, a certain quantity of silicon remains in solution as monosilicic acid (H4SiO4), and it is now believed that this fraction is responsible for the prophylactic properties of silicon by modulating the plant's defence response. The beneficial effects of silicon on the incidence and the intensity of fungal diseases were described in a variety of pathosystems. However, the exact nature of the interaction between silicon and plant defence or resistance mechanisms is unknown. We propose that silicon influences the biochemical activity of components responsible for post-elicitation intracellular signaling, and that it thus has a bioactive role in the defence or the resistance of plants against pathogenic fungi. In order to study the biological and prophylactic effects of silicon treatment, and using the oligonucleotide microarray technology, we carried out a complete transcriptome analysis of Arabidopsis plants inoculated or not with powdery mildew (Erysiphe cichoracearum) and treated or not with silicon. The application of silicon to non-inoculated plants significantly altered the relative abundance of only two out of ca. 40,000 transcripts (28,500 genes and over 10,000 unannotated transcripts). On the other hand, the inoculation with Erysiphe cichoracearum resulted in the significant induction of 2,387 genes and to the repression of 1,583 genes. For each gene and using innovative data-processing methods, we generated a single simplified functional annotation by using the final annotation of the Arabidopsis genome, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Aracyc biochemical pathways and two public databases of transcription factors (DATF and ATTFDB). The differentially expressed genes were classified into 58 functional categories including 24 categories directly involved in plant defence mechanisms and pathogenesis. This approach made it possible to appreciate as a whole the cascade of transcriptional events involved in the interaction Arabidopsis-powdery mildew in presence or absence of silicon. In response to the infection, 73% of defence genes (R genes, stress-related transcription factors, genes responsible for the generation of stress signals, the synthesis of antimicrobial compounds, metabolism of reactive oxygen species, PR proteins, etc.) were significantly induced. On the other hand, the disease critically affected essential processes such as photosynthesis and energy metabolism. Silicon treatment resulted in a 25% median attenuation of the repression in infected plants, whereas induction was maintained to a similar level in control and silicon treated plants. The role of silicon in plant biology, its essential or beneficial status, its direct or indirect effect on growth, and its bioactive or mechanical mode of action are discussed for decades. Results obtained in this study lead us to conclude that silicon alone does not have an effect on the metabolism of plants growing in a controlled environment (i.e. without stress), which supports the assumption that silicon is not an essential element. Silicon treatment increases the resistance to fungal infection, which results in an attenuation of the repression of genes involved in the energy metabolism and photosynthesis; silicon is thus beneficial for a plant under biotic stress. The mode of action of silicon in prophylaxis and stress attenuation seems active because gene induction by the fungal pathogen Erysiphe cichoracearum is maintained in silicon treated Arabidopsis; the influence of silicon thus cannot be reduced to a mechanical effect.

Doctor of Philosophy / Department of Biology / Ari M. Jumpponen / Dark septate endophytes (DSE) are darkly pigmented microfungal ascomycetes commonly observed in the healthy plant roots. Studying the functional roles of DSE is challenging as fundamental information about their identity, nutritional requirements, host range or host preference are lacking. Objective 1: root colonizing fungi were isolated from Konza plants roots and DSE fungi were identified by testing Koch’s postulates using leek plants. Periconia macrospinosa and Microdochium sp., were identified as DSE as they produced microsclerotia and chlamydospores in the root cortex. Select DSE were tested for their enzymatic capabilities and ability to utilize nitrogen sources: fungi tested positive for amylase, cellulase, polyphenol oxidases and gelatinase. Periconia isolates utilized organic and inorganic nitrogen suggesting facultative biotrophic and saprotrophic habits. Objective 2: a Microdochium isolate and three Periconia isolates were screened on 16 plant species (six native grasses and forbs, four crops) in a resynthesis system to test host range. DSE colonized all plant species, albeit to varying degrees. Host biomass and nutritional levels to DSE colonization varied within and among host species confirming the broad host range. Based on % responsiveness to DSE colonization, a metric similar to ‘mycorrhizal dependency’, grasses responded positively, while forbs and crops responded negatively. To test this observed ‘host preference’ under natural conditions, Konza roots from seven grass and nine forb species were surveyed for DSE colonization. Grasses hosted 50% greater DSE than forbs, supporting the broad host range and host preference of DSE fungi. Objective 3: three conspecific Arabidopsis ecotypes, Col-0, Cvi-0 and Kin-1 were inoculated with 25 P. macrospinosa isolates in resynthesis system. The three ecotypes responded differently to inoculation: Col-0 and Cvi-0 responded negatively, while Kin-1 response was neutral. Despite the negative or neutral response, each ecotype responded positively to one or two isolates. The outcomes were along the mutualism-parasitism continuum precluding an unambiguous assignment to any particular life-style. This study shows that the outcomes along this continuum are dictated by host and fungal genotypes. However, the more important question about their function remains. Additional studies with Arabidopsis microarrays are likely to provide unique insights into the potential roles of DSE.

Doctor of Philosophy / Department of Biology / Mark Ungerer / Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Using the model plant species Arabidopsis thaliana, I studied the intra-specific variation in freezing tolerance among natural accessions across its native range. Considerable variation in freezing tolerance among 71 selected accessions was observed both with and without a prior cold acclimation treatment, suggesting that both differences in cold-acclimation capacity and in intrinsic physiology contribute to this variation. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of these accessions. This clinal pattern of variation is found to be attributable, at least in part, to relaxed purifying selection on CBF/DREB1 genes in the species’ southern range. These CBF/DREB1 genes encode transcriptional activators that play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit significantly higher levels of nonsynonymous polymorphisms in coding regions of CBF/DREB1 genes. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in mutations in regulatory regions that lead to abrogated expression. These mutations in coding and regulatory regions compromise the function of CBF/DREB1 transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. These mutations could be selective neutral or beneficial in southern accessions depending on whether there is an allocation cost associated with cold acclimation. The fitness benefit and possible allocation cost of cold acclimation was examined in freezing and freezing-free environments using natural accessions exhibiting contrasting abilities of cold acclimation as well as transgenic CBF gene over-expression or knockdown/knockout lines. The extent to which cold acclimation benefits the plant in presence of freezing temperature is revealed, but a cost of cold acclimation wasn’t detected in the absence of freezing temperature under our experimental design, which suggests that these mutations in CBF genes in southern accessions might be neutral to natural selection.

Chez les végétaux, la mort cellulaire programmée (MCP) permet d'assurer le développement optimal de la plante et de protéger l'organisme contre différents stress biotiques et abiotiques. Peu d'effecteurs de la MCP végétale sont connus et caractérisés. L'objectif de ce projet de maîtrise était donc d'améliorer nos connaissances sur l'un de ces effecteurs : la protéine anti-apoptotique Bax inhibitor-1 (BI-1). Chez les plantes, BI-1 possède un rôle de protection contre la MCP induite par différents stress et ce projet s'est intéressé à la caractérisation de ce rôle pour trois agents stressants. L'utilisation de différents mutants nuls et surexpresseurs d'AtBI-1 chez la plante modèle Arabidopsis thaliana pour des tests de germination et l'extraction de la chlorophylle a permis la démonstration d'un rôle de protection d'AtBI-1 contre la MCP induite par les UV-C et le méthyl viologène puisque son absence augmente la sensibilité des plantules. Par contre, dans les conditions utilisées, AtBI-1 ne semble pas jouer de rôle de protection contre une MCP induite par les cytokinines. La suite du projet s'est concentrée sur la régulation du gène AtBI-1 suite à une irradiation aux UV-C. Une région régulatrice spécifique aux UV-C a pu être identifiée grâce à une série de constructions comprenant la région régulatrice d'AtBI-1 coupée à différents sites de restriction et couplée au gène rapporteur GUS. Cette région régulatrice est probablement impliquée dans la régulation négative du gène puisque sa délétion entraîne une forte augmentation de l'expression basale d'AtBI-1. Enfin, une analyse bio-informatique de cette région régulatrice spécifique aux UV-C a permis l'identification de plusieurs motifs et sites de liaison pouvant potentiellement être impliqués dans la régulation d'AtBI-1 par les UV-C. De plus, des essais de gel à retardement sur cette région régulatrice montrent qu'elle est liée par un facteur nucléaire en conditions normales et qu'il y a perte de cette liaison suite à un traitement aux UV-C. L'identification de ce facteur nucléaire permettra de déterminer son implication dans la régulation d' AtBI-1 par les UV-C.

La réussite de la mise en place d’une résistance efficace chez la plante hôte requiert la présence d’un dialogue entre les différentes voies signalétiques menant à l’induction de la défense. L’implication de l’extinction génique post-transcriptionnelle dans la résistance contre des agents pathogènes, autres que les virus, est une nouvelle avenue qui va permettre de disséquer le lien qui peut exister entre les deux voies immunitaires de la plante : celle qui est déjà importante pour lutter contre les bactéries et les champignons (voie induite à la suite de la reconnaissance des éliciteurs) et celle de l’extinction génique post-transcriptionnelle. Les résultats que nous avons obtenus ont une signification très importante concernant l’implication de l’extinction génique post-transcriptionnelle dans la résistance des plantes contre les agents pathogènes d’une manière générale et Botrytis cinerea, Alternaria brassicicola et Pseudomonas syringae en particulier. Mon travail de recherche a permis d’identifier le miR472 comme étant un acteur qui favorise la mise en place de la maladie causée par des agents pathogènes du type nécrotrophe et hémibiotrophe. De plus j’ai pu, en utilisant le modèle P. syringae pv tomato DC3000-Arabidopsis, disséquer dans le détail, comment la bactérie manipule la machinerie de la plante pour activer la voie des microARNs menant à l’accumulation du miR472, responsable de la suppression de la résistance basale et du PTI. Cette suppression passe par l’inhibition de sa cible, la protéine NB-LRR; RDS1 un nouveau régulateur impliqué dans le PTI induit par la Flg22. Cette étude permettra une avancée remarquable dans les connaissances reliées à l’immunité des plantes, ce qui constituerait une étape clé dans la découverte de nouvelles stratégies de lutte contre les agents phytopathogènes. Sur le plan fondamental, cette étude a permis de consolider le dialogue qui existe entre les voies immunes de la plante et de mettre en évidence un nouvel acteur impliqué dans la résistance basale. En matière d’application, comprendre les mécanismes de défense et de résistance des plantes aux agents pathogènes est primordial pour développer des plantes génétiquement résistantes et des stimulateurs de défense naturelle (Vaccin de plantes). Ceci constitue un enjeu majeur dans l'objectif de concevoir de nouveaux moyens de lutte phytosanitaire, à la fois plus respectueux de l’environnement et ayant moins d’impact sur la santé humaine.

Growth rate and flowering time are agriculturally important traits that are linked to fitness, productivity and reproductive success of plants. To study the genetic basis for natural variation in growth rate and flowering time between local accessions of Arabidopsis thaliana, hybrids were produced between fast growing / late flowering and slow growing / early flowering parents. F3 and F5 hybrid families were grown under a range of conditions – under a constant controlled environment, outside over the winter and outside in spring and early summer. Growth rates were estimated from repeated images of rosettes. Flowering time, as number of leaves to flower, was also recorded both in control and natural conditions for F5 lines. Damage by slugs and stress-induced production of anthocyanin pigments were also recorded for plants grown outside. Broad-sense heritability estimates were higher for F5 families than F3, in which more loci will segregate, and ranged from 48% to 89%. No significant correlation between growth rates under different environments was observed in most cases for F3 populations, however significant correlations were detected for F5 families outside and under controlled conditions, suggesting that same genes can affect growth rate in more than one environment. The genotypes of F3 families were determined at thirty-nine SSLP (simple sequence length polymorphism) loci and used in regression with phenotype data to search for quantitative trait loci (QTL). Significant QTLs were detected in F3 families for growth rate, flowering time and anthocyanin production, but not for herbivore damage. To confirm QTL detected in the F3 and to detect additional loci, bulk segregant analysis was carried out in F5 families grown under different conditions. Potentially linked markers were tested further in individual F5 plants and QTL mapped on a finer scale in F5 families that remained heterozygous for candidate regions. VIP5 and LDL1 were selected as potential candidate genes for flowering time variation. These genes were sequenced for two parental alleles. A transposon insertion and 5’ UTR deletion were found in the LDL1 allele from the late flowering parent and SNPs (single nucleotide polymorphisms) were observed throughout the gene. However both alleles appeared to be expressed at similar levels. Transgenic lines have been produced carrying the LDL1 allele from the early flowering parent (4D1) in the background of the later flowering parent (11C1). This work is on-going and will hopefully reveal whether LDL1 underlies differences in flowering behaviour seen between 11C1 and 4D1.

Widespread antibiotic resistance is a major public health concern, and plants represent an emerging antibiotic exposure route. Recent studies indicate that crop plants fertilized with antibiotic-laden animal manure accumulate antibiotics, however, the molecular mechanisms of antibiotic entry and subcellular partitioning within plant cells remain unknown. Here we report that mutations in the Arabidopsis locus Multiple Antibiotic Resistance (MAR1) confer resistance, while MAR1 overexpression causes hypersensitivity to multiple aminoglycoside antibiotics. Resistance is highly specific for aminoglycosides and does not extend to antibiotics of other classes, including the aminocyclitol, spectinomycin. Yeast expressing MAR1 are hypersensitive to the aminoglycoside, G418, but not to chloramphenicol or cycloheximide. MAR1 encodes a protein with 11 putative transmembrane domains with low similarity to ferroportin1 from Danio rerio. A MAR1:YFP fusion protein localizes to the chloroplast, and chloroplasts from plants overexpressing MAR1 accumulate more of the aminoglycoside, gentamicin, while mar1-1 mutant chloroplasts accumulate less than wild type. MAR1 overexpression lines are slightly chlorotic, and this chlorosis is rescued by application of exogenous iron. MAR1 expression is also downregulated by low iron. Taken together, these data suggest that MAR1 is a plastid transporter that is likely to be involved in cellular iron homeostasis, and allows opportunistic entry of multiple antibiotics into the chloroplast. mar1 mutants represent an interesting example of plant antibiotic resistance that is based on the restriction of antibiotic entry into a subcellular compartment. Knowledge about this process – and other processes of antibiotic entry – could enable the production of crop plants that are incapable of antibiotic accumulation, aid in development of phytoremediation strategies for decontamination of water and soils polluted with antibiotics, and further the development of new plant-based molecular markers. The work described here also contributes to our understanding of how plants interact with the antibiotics they encounter, both in the laboratory (where aminoglycosides such as kanamycin are used heavily to select for transgenics) and in the natural environment. / text

The eATP pathway begins a cascade of events which includes the involvement of nitric oxide synthase (NOS) and nitrate reductase (NR) in the production of nitric oxide (NO). Research has shown that SNAP (S-nitroso-N-acetylpenicillamine) and NONOates (diazeniumdiolates) promote the availability of NO and, with the addition of guanylate cyclase, form cyclic guanine monophosphate (cGMP), and root hair growth is promoted. Phosphodiesterases (PDE) break down the cGMP and agents such as IBMX and Viagra inhibit the PDEs thereby inhibiting root hair growth. Several questions remain to be answered. How much cGMP is necessary for the promotion of root hair growth? Is there an optimal concentration of cGMP which stimulates root hair growth, above which is inhibitory, or below which is ineffective? Is there a “non-hydrolyzable analog” of cGMP which is more effective at promoting root hair growth? Is it possible to see inhibition of root hair growth with exposure to a known inhibitor, such as ATPγS, and then reverse the inhibition with a “non-hydrolyzable analog” of cGMP? Answering these vi questions is the substance of this research and the answers will provide direction and understanding to the growth-promoting and regulatory role eATP plays in signal transduction pathways in plants. With the hypothesis asking whether the effects of NO on root hair growth is cGMP-dependent or cGMP-independent we found that there is no consistent concentration of non-hydrolyzable cGMP analog which promotes root hair growth. Additionally we found that the 8-Br-cGMP analog promotes root hair growth more consistently in Arabidopsis thaliana than its counterpart, dibutyryl cGMP. We substantiated previously published results showing an inhibition of root hair growth when root hairs were exposed to high concentrations of ATPγS. Based on these results we believe the promotion of root hair growth in Arabidopsis thaliana to be mediated independently of cGMP. / text

Conversion of aspartate and asparagine residues to isoaspartate is a prevalent covalent protein modification in cells. The accumulation of these altered residues can lead to the loss of protein function and the consequent loss of cellular function. The L-ISOASPARTATE METHYLTRANSFERASE (EC 2.1.1.77) (PIMT) iteratively methylates abnormal isoaspartyl residues leading to conversion to L-aspartate, thereby mitigating the injurious effects of aging. Arabidopsis thaliana is unique among eukaryotes studied to date in that it possesses two genes (At3g48330 (PIMT1) and At5g50240 (PIMT2)) encoding PIMT. The PIMT2 gene exhibits a complex transcriptional control involving different transcriptional initiation sites and 5'- and 3'- alternative splice site selection in the first intron. Varying the transcriptional initiation site results in alternative targeting of the PIMT2 proteins thus produced to: 1) the nucleus, or 2) the cytoplasm, while PIMT1 is cytosolic. Inclusion of a 51 nucleotide 5 alternatively spliced sequence with or without a nine nucleotide 3 alternatively spliced sequence dramatically alteres the subcellular protein localization from the cytoplasm and around the chloroplast to inside the chloroplast. All recombinant PIMT2 isoform tested exhibit PIMT activity, although solubility varied among them. Multiplex RT-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to -TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous ABA. Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA application or applied stress presumably through an ABA-dependent pathway. Furthermore, Cleaved Amplified Polymorphic Sequence analysis of the PIMT2 amplicons has shown that the ratio among the splicing variants alters upon ABA application, implicating a role for the spliceosome or differential RNA stability in orchestrating the plant's response to stress. T-DNA insertional mutants of both genes were isolated but no obvious phenotype has been identified. The double mutant has been generated and will be evaluated.

Poly(A) tail addition to pre-mRNAs is a highly coordinated and essential step in mRNA maturation involving multiple cis- and trans-acting factors. The trans-acting factor, poly(A) polymerase (PAP) plays an essential role in the polyadenylation of mRNA precursors. The Arabidopsis thaliana genome contains four putative PAP genes. We have found, using in silico analysis and transgenic plants expressing GUS under the control of the four PAP promoters, that each of these genes is expressed in overlapping, yet unique patterns. This gives rise to the possibility that these genes are not redundant and may be essential for plant survival. To further test this, inducible RNAi and T-DNA mutagenized plants were obtained and analyzed. Plants lacking all, or most, of each PAP gene product, due to RNAi induction, were not viable at any of the stages of plant growth tested. Furthermore, T-DNA PCR analysis determined that no plants containing a homozygous mutation, were viable. This data reveals that lack of any of the four PAP gene products has a significant effect on the plants ability of survive, thus indicating that each PAP gene is essential. Finally, transient expression experiments with each of the full length PAP cDNAs fused to GFP showed that the PAP I, PAP II and PAP IV gene products are localized throughout the nucleus and within nuclear speckles. The cellular localization of PAP III could not be determined.