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The transition to flowering is an important decision for plants since seed-setting and the survival of the progeny depend on the environmental conditions prevalent during this transition. Therefore, to ensure maximum reproductive success, plants have evolved several regulatory mechanisms to enable them flower at the most appropriate time. Environmental parameters such as light, temperature and nutrient availability as well as endogenous factors such as age and hormonal status of the plant profoundly affect floral transition (Boss et al., 2004; Srikanth and Schmid, 2011). Studies in Arabidopsis and other model plant species have identified several distinct genetic pathways that integrate the information from the endogenous and environmental cues to regulate flowering (Boss et al., 2004; Srikanth and Schmid, 2011). Many components and gene regulatory networks identified in Arabidopsis are conserved in other commercially important species including rice, maize, sorghum, potato and tomato. Therefore, it is important to understand the basic mechanisms that modulate the flowering response in model plants such as Arabidopsis thaliana, the knowledge from which can be used to develop better adapted and high-yielding varieties of crop plants in the wake of challenges like global warming and increasing food demand. In the present study, we have studied the function of VLB1 and VLB2, genes that code for plant-specific Zn-finger transcription factors. Previous studies from our laboratory (Pratibha Choudhary, Ph.D thesis, 2011) and by other research groups have reported that VLBs redundantly promote flowering in A. thaliana (Yasui et al., 2012; Celesnik et al., 2013). However, the underlying mechanism of this regulation is not well understood. Our data suggests that VLBs redundantly promote the transition to flowering specifically in the photoperiod pathway, the major floral induction pathway in A. thaliana. CO, which is the 93 key regulatory gene in this pathway, is regulated by various factors at the transcriptional as well as post-transcriptional level (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002; Srikanth and Schmid, 2011). Using genetics, we show that VLBs and CO function together to promote flowering in the photoperiod pathway. Further, our BiFC results reveal that VLBs and CO interact physically. Nevertheless, the physical interaction between VLBs and CO needs to be further validated by in vitro and in vivo by co-immunoprecipitation experiments. We hypothesize that the interaction between VLBs and CO is important to regulate FT expression and hence, flowering. However, whether VLBs interact with CO and promote the CO-stability, or facilitates its recruitment to the FT promoter region, still needs to be determined. Apart from its role in flowering, VLBs have been recently shown to regulate biotic and abiotic responses in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Also, even though it has been demonstrated that VLBs code for transcription factors, no direct targets of VLBs have been reported till date. We performed a whole genome trancriptome-profiling and found that several important classes of genes including WRKY, RLPs, NBS-LRR and JAZs were affected suggesting that, in addition to their role in floral transition, VLBs have important functions in other plant processes as well. In fact, vlb1vlb2 mutant showed an early senescence phenotype and many senescence-associated genes (SAGs) were up-regulated in our microarray experiments, which was further validated by qRT-PCR analysis. By comparing the differentially-regulated genes and PatMatch analysis, we have identified 82 putative direct targets of VLBs in the Arabidopsis genome which need to be validated by chromatin immunoprecipitation (ChIP) assay and functional studies. 94 Results of global transcriptome analysis revealed that the expression of several JA-signaling and response genes was significantly down-regulated. JA is an important phytohormone involved in plant defense and other developmental processes such as stamen development, root growth and senescence (Wasternack, 2007). Results from the JA-induced expression analysis and root inhibition assay confirmed that JA-signaling and response are indeed compromised in the vlb1vlb2 double mutant. Moreover, in vitro DNA-binding assay showed that MYC2, the key transcriptional regulator of JA-responsive gene expression, is a direct transcriptional target of VLB2. A recent study reported that loss-of-function of VLB genes impairs plant defense while their overexpression confers biotic stress tolerance in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Compromised JA signaling in the vlb1vlb2 double mutant might partly explain this reduced tolerance to pathogens. However, whether VLBs are associated with the MYC2 promoter in planta needs to be tested by performing ChIP and other in vivo assays. In conclusion, our study shows that VLBs have important regulatory roles in diverse processes including control of flowering time, senescence and JA signaling in Arabidopsis. The validation and functional characterization of the direct targets of VLBs will shed more light on the role of VLBs. Since VLBs are conserved in vascular plants, it will be interesting to see if the function of VLBs is also conserved across species and what might be its ancestral function in evolution.

Stomatal development and its regulation by environmental conditions (light, CO2 concentration) and physiological processes in the leaf of higher plants were investigated. The study was based on the assumptions that stomatal development should be regulated by signals coming from both external environment and leaf interior, and that the signal should be transduced from cotyledons to leaves. Transgenerational effect in stomatal development was also studied. Molecular and physiological approaches were applied to reveal the relationship between leaf environment, stomatal development, stomatal function and leaf physiology.

In order to identify new players in fruit development, a forward genetic screen was performed on EMS mutagenized plants. A mutant named elongated gynophore (egy) was identified in the M2 population based on altered fruit morphology. Genetic analysis established that the egy phenotype is due to a monogenic and recessive mutation. The egy plants show additional developmental defects including shorter root, narrower cotyledons and malformed leaf lamina. Molecular mapping and whole genome sequencing analyses showed a G/C deletion at the position 4414980 on the AT5G13680 gene locus which is predicted to encode the ELONGATA2 (ELO2) protein. ELO2 is a constituent member of the elongator complex which helps in transcriptional elongation in association with the phosphorylated form of RNA polymerase II. This complex has been implicated in controlling development, abiotic stress and biotic stress. Genetic complementation test confirmed that egy is indeed allelic to elo2-3. Surprisingly, the EGY overexpression line 35S::EGY showed loss-of-function phenotype, suggesting transgene silencing. In angiosperms, fruit is derived from the fertilized ovary. The initiation of the female reproductive organ commences with a lump of cells which eventually develops into the gynoecium with a stigma, a style, two fused ovaries and a gynophore, arranged from the apical to basal axis in that order. Genetic networks faithfully shapes up the carpel primordium into predetermined gynoecium shape. Following fertilization, siliques elongate concomitantly with developing embryos. Here we show that the egy mutant has apical basal patterning defect with longer gynophore at the base. This gynophore phenotype resembles the phenotype found in the mutants with altered auxin and cytokinin levels/signaling. We show that egy is hypersensitive to cytokinin treatment; egy fruits treated with cytokinin display phenotype similar to the plants expressing IPT7 under fruit-specific promoter. These results suggest that broadened shoulders at the apical region of egy gynoecium possibly results from higher cytokinin level/response. Genetic interaction studies have shown that EGY act independent of AGAMOUS and PEAPOD to suppress the medio-lateral growth of the apical gynoecium region. Genetic and expression studies suggest that PINOID and TMO5/T5L1 work downstream to EGY, while ETTIN acts in parallel to EGY. We also observed larger seeds in the egy mutant and show that this is controlled maternally. Thus, the gametic lethality in egy can possibly be accounted for by the defective ovules. We show that egy primary roots are shorter compared to Col-0, though egy seeds have longer embryonic root to begin with, suggesting a defect in cell division. The root cells are arranged radially in a stereotypic pattern in root meristem from the outer epidermis to the inner stele specific the vascular bundles. The four QC cells are also surrounded by stem cells of various identities. This stereotypic pattern of cell arrangement is perturbed in the egy root. The stele, composed of pericycle and vascular bundles is reduced in the egy mutant, suggesting a positive role of EGY in vascular cell division. Confocal microscopic studies and real-time PCR data suggest that TMO5/T5L1 work downstream to EGY. Thus, the Arabidopsis „ELONGATOR‟ complex regulates the transcription of target genes that are necessary for plant growth and development. A proposed genetic network for the role of EGY in fruit and root development. Based on the genetic interaction studies and expression analysis, we have placed EGY in the existing molecular network that control fruit and root vascular development in Arabidopsis

L’infection virale compatible des plantes est une cause majeure des pertes de rendements des récoltes. Nousavons analysé, suite à une infection par les tobamovirus, les réponses de l’hôte au niveau de la régulation del’expression des gènes ainsi qu’au niveau des mécanismes moléculaires déterminant l'infection. Des plantesd'Arabidopsis infectées par le Virus de la mosaïque du colza (ORMV) ont été utilisées pour étudier leschangements d’expression des gènes induits par le virus. Conformément à l'expression d'un suppresseur desilencing par le virus, les forts changements de populations d’ARNs ne corrèlent pas d’une façon significativeavec les changements de taux d’ARNm cibles. En se concentrant sur les altérations des processusphysiologiques, deux gènes potentiellement impliqués dans les réponses à l'infection virale ont étéfonctionnellement caractérisés. Le premier gène code pour CIPK12. L’ARNm issu de ce gène montre uneaccumulation suite à une infection par ORMV. De plus, l'expression transitoire de CIPK12 dans des feuilles deN. benthamiana semble inhiber la propagation de l’ORMV dans cet hôte. La deuxième protéine étudiée dansce travail est CDC48B qui s'accumule suite à une infection tobamovirale et agit dans la maintenance desmembranes du réticulum endoplasmique (RE). De plus, CDC48B interagit avec la MP virale qui s’accumuledans des inclusions associées aux membranes du RE en vue d’améliorer son extraction des membranes et salocalisation au niveau des microtubules et dans le cytosol. Ainsi, deux protéines induites par l’infection viraleavec un potentiel d'interagir avec la MP virale et d'influer sur son activité lors de l'infection, sont décrites danscette thèse. / Infection of crop plants with compatible viruses is a major cause of losses in harvest yield. Here, we analyzedhost responses to tobamovirus infection at the level of gene regulation and expression, and at the level ofmolecular mechanisms determining the outcome of infection. ORMV-infected Arabidopsis plants were used toaddress virus-induced changes in gene expression. Consistent with the expression of a silencing suppressor bythe virus, strong changes in sRNA populations in virus-infected plants are not significantly correlated withcorresponding changes in the levels of the corresponding mRNA targets. Focusing on altered physiologicalprocesses, two genes with potential involvement in responses to virus infection were functionallycharacterized. The first gene encodes CIPK12, a (CBL)-interacting protein kinase, involved in decodingcalcium signatures. The mRNA of this gene accumulates upon ORMV infection and further observationsprovide evidence indicating that the regulation of the CIPK12 mRNA level occurs at the post-transcriptionallevel. Transient expression of CIPK12 in N. benthamiana leaves appears to inhibit the cell-to-cell spread ofORMV in this host. The second protein addressed in this work is CDC48B. CDC48B accumulates upontobamoviral infection and functions in ER-membrane maintenance. Consistent with this function it interactswith virus-accumulated MP in ER-associated inclusions to promote its extraction form the membrane and itslocalization to the cytosol. Thus, in this thesis I describe two virus-induced proteins with potential to interactwith viral MP and to influence the subcellular localization of this protein during infection.

Abstract The timing growth and reproduction are critical to the fitness of plants and animals. The timing also has an important role in local adaptation. Locally adapted plants may have different responses to photoperiod and other environmental cues and genes or alleles behind underlying differences may differ between populations. The molecular genetics and physiology of flowering of the plant molecular biology model organism Arabidopsis thaliana is being intensively studied, and this offers a good opportunity to study the genetic basis of flowering time variation in related non-model species. The closely related perennial species Arabidopsis lyrata provides an interesting comparison to A. thaliana because of its different ecology, mating system and life history. The influence of sampling designs on clustering methods was analyzed using simulations and microsatellite data in the selfing A. thaliana. It was found that sample size has a large effect on the resulting number of clusters and sampling too few individuals per locality could lead to a severe underestimation of the real number of subpopulations. Patterns of sequence variation in flowering time genes and association between polymorphisms at FRI (and FLC) and flowering time was studied in A. thaliana and in A. lyrata to find out whether the genes were responsible for flowering time differences between and within natural populations. In A. thaliana there was no significant association between polymorphisms at FLC and FRI and flowering time. In A. lyrata the FRI gene was polymorphic for an indel associated with flowering time variation within two Northern European populations, suggesting that the indel (or a linked polymorphism) was involved in flowering time variation. However, FRI did not explain the flowering time differences between A. lyrata populations, and other loci must be involved. Patterns of diversity and divergence at flowering time related loci were compared against a set of random reference loci to examine the roles of selection and demography. Sequence variation in the studied A. lyrata populations departed from the standard neutral equilibrium model and it has been influenced by recent historical events, most likely bottlenecks. The level of silent and synonymous polymorphisms in flowering time genes was highly reduced and this can be likely explained by selective sweeps at flowering time genes. / Tiivistelmä Kasveilla kukkimisen ajoittaminen suotuisaan ajankohtaan on hyvin tärkeää suvullisen lisääntymisen kannalta. Kukkimisen oikealla ajoituksella on myös tärkeä rooli kasvien sopeutumisessa paikallisiin olosuhteisiin. Kukkimisaikamuunteluun vaikuttavat useimmiten lukuisat geenit sekä ympäristötekijät, jotka voivat vaihdella alueellisesti ja populaatioiden välillä. Vaikka kukkimiseen ja kukkimisaikaan vaikuttavia tekijöitä tunnetaan jo hyvin, luonnonpopulaatioiden muuntelun ja paikallisen sopeutumisen geneettinen tausta on huonommin tunnettu. Väitöstutkimus keskittyy Arabidopsis-populaatioiden paikalliseen sopeutumiseen tarkastelemalla kukkimisajan muuntelua ja siihen vaikuttavia geeneettisiä tekijöitä. Tutkimuksessa käytetyt geneettiset aineistot perustuvat osin neutraaleihin merkkigeeneihin (mikrosatelliittimuunteluun), ja osin sekvenssien nukleotidimuunteluun. Väitöstutkimuksessa on simulointien avulla selvitetty populaatiosta analysoitavien yksilöiden lukumäärän merkitystä populaatiorakenteen selvittämisessä itsesiittoisella lituruoholla (Arabidopsis thaliana). Tulosten mukaan on hyvä analysoida useampia yksilöitä paikallisista populaatioista, sillä liian pienet otoskoot voivat johtaa ryhmien määrän aliarvioimiseen. Koalesenssisimulaatiot osoittavat idänpitkäpalon (Arabidopsis lyrata) populaatioiden poikkeavan tasapainotilasta ja populaatioissa tapahtuneen populaatiokoon muutoksia (ns. pullonkaulailmiö). Tutkimuksessa havaittiin sekvenssimuuntelun olevan alhaisempaa kukkimisaikageeneissä kuin referenssigeeneissä todennäköisesti positiivisen valinnan vaikutuksesta. Tutkimuksessa todettiin, että FRI geenissä tapahtuneet mutaatiot ovat kahdessa tutkitussa lajissa erilaisia luonteeltaan, mutta geenillä on kuitenkin samanlainen rooli kukkimisajan määräämisessä. Assosiaatiokokeissa lituruoholla ei Pohjois-Euroopan populaatioissa löydetty merkitsevää assosiaatiota FRI geenin ja kukkimisajan välillä, kun puolestaa idänpitkäpalolla FRI vaikutti kukkimisaikamuunteluun kahdessa pohjoisessa populaatiossa.

M.Sc. (Biochemistry) / Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.

Several mechanisms have been proposed to explain the process of vein pattern formation in plant tissues. The most widely accepted amongst biologists is the canalization hypothesis, derived from pea root and stem experiments. According to this hypothesis, a signal, thought to be the phytohormone auxin, is transported polarly from cell to cell from the shoot to the root and is canalized progressively into narrow channels of high auxin fluxes that later differentiate to become vascular tissue. In this project, we set out to test whether auxin canalization drives vein pattern formation, using Arabidopsis thaliana mutants with increased auxin transport (max4-1, max3-9, max2-1 and max1-1). We predicted that the mutants would have distinct vein patterns and especially different angles between the primary and secondary veins, compared to the wild type. First rosette leaves of 15 plants per genotype were harvested for analysis each day from 7 to 17 days after sowing, giving a total of eight hundred twenty-five leaf samples to analyze. Venation patterns were extracted and analyzed using custom-made software written with Matlab. Overall, compared with the wild type, mutants with the highest auxin transport (max4-1 and max3-9) had different vein patterns at early developmental stages, confirming a role for auxin transport in vein patterning. However, veins of mutants and wild type connected at similar angles, which is not consistent with the auxin canalization hypothesis, as originally formulated.

M.Sc. / Since plants are stationary, their immune systems have adapted to their environments to enable them to overcome or respond appropriately to various environmental, physical and physiological stresses that they may encounter by developing complicated defense mechanisms. The plant defense response activates complex biochemical and structural changes in plant cells. Heat stress per se, appears to be a priority stress response in plants, and increased disease susceptibility may be a result of this response. In this study, altered gene expression levels mediated by a heat treatment in Arabidopsis thaliana seedlings were analyzed. Seedlings were exposed to a heat stress of 42C for 30 minutes, followed by a 2.5 hour recovery period at 25ºC. RNA that was isolated from the heat stress treated plants as well as control plants (untreated) was used to perform suppression subtractive hybridization (SSH) in order to obtain a forward and a reverse DNA library. The forward SSH library represented the genes that were up-regulated due to the heat shock and the reverse SSH library represented the down-regulated genes. Sequenced clones from these libraries were BLAST against the A. thaliana genome using the Genbank database and the Accession numbers retrieved were then used for Bioinformatics analysis to obtain functionality of the genes found. The bioinformatics tools used were TAIR tools, DAG graphs and FatiGO and genes were categorized into the biological processes, molecular functions and cellular components. The TAIR tools and FatiGO were then used to analyze microarray data obtained for a similar study, in order to compare the genes identified with SSH. The genes coding for photosystem IID, serine-type peptidase, phospholipase D α, a S-locus protein kinase, regulator of chromosome condensation (RCC1) and Glucose-6-phosphate translocator are prominently up-regulated whilst other genes encoding photosystem I, plastocyanin-like mavicyanin, carbohydrate trans-membrane transporter MSS1, zinc finger C3HC4 ring family protein, ubiquitin conjugating enzyme 35 (UBC35) and integral membrane family proteins are significantly down-regulated. The FatiGO results helped to assign functionality to the genes that were found. For the SSH forward library, the cellular protein metabolic pathway was the most highly expressed term (19.21%), whereas in the microarray data, the term „positive regulation of response to stimulus‟ and membrane disassembly had a 100% expression. The reverse SSH data (down-regulation) found phosphate metabolic process as the most highly expressed term with an expression of 44.36% ix and the microarray data (negative fold-change) found the term photorespiration to be the most highly expressed with 93.54% expression. These high levels of negative expression indicate the down-regulation of these processes in the cell during heat shock. From these results it can be assumed that at the onset of a heat stress, the plant‟s immediate response is to activate pathways of regulation as a response to the stimulus as a self-protection mechanism, and repress other pathways such as photorespiration in order to preserve its energy such as ATP. These findings suggest that the plant is well equipped to overcome stress in its environment by activation/repression of specific organelles and pathways in the system, in order to maintain its equilibrium. Studies such as these can prove to be helpful to solve the interesting question of how a plant overcomes various environmental stresses in order to prevent disease susceptibility.

Les transporteurs ABC sont les membres d'une superfamille de protéines qui utilisent l'hydrolyse de l'ATP pour déplacer une large gamme de substrats au travers des membranes biologiques. Les membres de la sous famille ABCC sont généralement caractérisés par un domaine transmembranaire supplémentaire en région N-terminal (TMD0). Dans cette étude, nous avons analysé deux gènes ABCC de plantes : CrABCC1 de Catharanthus roseus et AtABCC13 son orthologue chez Arabidopsis thaliana. L'analyse phylogénétique répartit les ABCC de plantes dans 3 clades distinctes. Les clades I et II sont spécifiques aux plantes tandis que le clade III est le seul associant des ABCC humains et de plantes. Le criblage de la base de données a permis d'identifier 16 séquences ABCC chez Catharanthus roseus parmi lesquelles 2 appartiennent à CrABCC. / ABC transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrate across biological membranes. Members of C. subfamily (ABCC) are generally structurally characterized by an additional (N-Terminal) transmembrane domain (TMD0). In this study the analysed two plant ABCC : CrABCC1 from Catharanthus roseus and AtABCC13, it's ortholog in Arabidopsis thaliana. Phylogenetic analysis of plant ABCCs separates their protein sequences over three distinct clusters : I and II are plant specific whereas cluster III is the only gathering humain and plant ABCCs. Screening of plant database allowed us to identify 16 different ABCCs sequences in Catharanthus roseus.