Phytocannabinoids are the active substances responsible for the medicinal and psychotropic effects of Cannabis sativa. Although the bioactivity of cannabis and its preparations have been known for millennia, several steps in the biosynthetic pathway leading to phytocannabinoids remain unclear. Phytocannabinoids are prenylated resorcylic acids which are formed in specialized plant organs called glandular trichomes. Following the analysis of a pre-generated cannabis trichome cDNA library, a type III polyketide synthase (tetraketide synthase; TKS) was identified and assayed, yielding three major compounds, hexanoyl triacetic acid lactone (HTAL), pentyl diacetic acid lactone (PDAL), and olivetol, yet no resorcylic acid was detected. This lack of resorcylic acid in enzyme assays has instigated the characterization of TKS and a search for putative cyclases in the cannabis trichome cDNA library, and involved protein pulldown, co-immunoprecipitation, and co-assay experiments. These experiments led to the discovery of a novel polyketide cyclase protein named olivetolic acid cyclase (OAC) responsible for the proper cyclization of a polyketide intermediate produced by TKS. This thesis shows that TKS assays conducted with OAC produce olivetolic acid (OA), an intermediate required during the biosynthesis of cannabinoids. The TKS/OAC spatial relationship was also investigated following the creation of fluorescent fusion proteins which show that the enzymes co-localized in vivo when viewed with confocal microscopy. Furthermore, yeast two-hybrid assays using TKS and OAC were performed to establish whether the enzymes physically interact. Finally, an attempt to determine the responsible amino acids involved in OAC’s mechanism was conducted by comparing the activity of single point OAC mutants with the wild-type OAC. Based on the available data, mechanisms for the production of HTAL, PDAL, olivetol, and OA are proposed.

Em estudos de proteção ao genoma contendo lesões de C. crescentus, foi feita uma varredura em uma biblioteca clones mutados pelo transposon Tn5 e sensíveis à luz UV-B e MMS. Dos 249 clones selecionados conseguimos identificar mutações em 102 genes, distribuídos em dez categorias funcionais, incluindo metabolismo de DNA. Além de genes já descritos como atuantes na defesa de genoma a lesões, os dados ainda adicionam funções potenciais para outros genes. Investigação mais detalhada indica que vários dos genes identificados podem afetar o estresse e ciclo celular, podendo estar relacionados a respostas do tipo pontos de checagem. Além disso, verificamos que mutantes para genes envolvidos em reparo excisão de nucleotídeos são mais sensíveis à H2O2, indicando que nessa bactéria, este sistema de reparo atua também em lesões oxidativas no DNA. Os dados obtidos são pioneiros no estabelecimento de funções para vários genes de C. crescentus e de outras alfa-proteobactérias. / This work aimed to identify genes related to DNA damage protection in C. crescentus, by means of screening a Tn5 -mutated library for clones sensitive to UV-B light and MMS. Out of 249 selected clones, we were able to identify mutations in 102 genes, classified in ten different functional categories, including DNA metabolism. In addition to genes already described as playing a role in genome defense to lesions, the results provide new potential functions to several other genes. More detailed investigation indicates also that the mutations in some of these genes may also affect cell stress and cell cycle, being potentially involved in check-point responses. Moreover, mutants for nucleotide excision repair genes were also found to be sensitive to H2O2, indicating that this DNA repair system also acts in DNA oxidative damage. These data are the first establishing a role in genome protection for several genes in C. crescentus, as well as other alpha-proteobacteria.

O choque frio em bactérias causa a indução de proteínas de choque frio de baixo peso molecular (CSPs), que desestabilizam estruturas secundárias do mRNA, permitindo sua tradução. Caulobacter crescentus possui quatro genes codificando CSPs: cspA e cspB são induzidos sob choque frio, e cspC e cspD, na fase estacionária. Neste trabalho, foi determinada uma nova seqüência para o gene cspC, revelando que a proteína CspC possui dois domínios CSD, como CspD. O mutante nulo para cspC apresentou sensibilidade em baixa temperatura e menor viabilidade em fase estacionária, com alterações na morfologia. A região regulatória foi mapeada por fusões de transcrição, e uma região ativadora da expressão foi identificada, mostrando uma regulação transcricional. Algumas condições nutricionais que disparam a indução do gene foram determinadas, indicando que sua expressão é influenciada pela ausência de glicose no meio, mas não pela ausência de nitrogênio. Este perfil de indução não depende da região ativadora, que, por sua vez, é necessária para os máximos níveis de expressão. / The cold shock response in bacteria involves the expression of cold shock proteins (CSPs), which destabilize secondary structures on mRNAs, allowing their translation. Caulobacter crescentus possesses four genes encoding CSPs: cspA and cspB are induced upon cold shock, while cspC and cspD are induced at stationary phase. In this work, a new sequence for the coding region of the cspC gene was determined, revealing that CspC contains two cold shock domains, like CspD. A null cspC mutant was sensitive to low temperature, presented reduced viability at stationary phase, and altered morphology. The regulatory region of cspC was mapped by transcriptional fusions, identifying a region responsible for activation of cspC expression, suggesting a transcriptional regulation. Some nutritional conditions triggering cspC induction were determined, indicating that its expression is influenced by glucose starvation, but not by nitrogen starvation. This expression profile was not dependent on the activation region, which, in turn, was required for maximum levels of expression.

Linhagens mutantes foram obtidas para os fatores sigma <font face=\"symbol\">sE (RpoE) e <font face=\"symbol\">sN (RpoN) da bactéria Xylella fastidiosa. O mutante rpoE mostrou-se sensível a etanol e a choque térmico. Análises de microarranjo de DNA, de RT-PCR quantitativo e mapeamento de sítios de início de transcrição permitiram definir o regulon <font face=\"symbol\">sE em resposta ao choque térmico. Verificou-se co-transcrição entre os genes que codificam para <font face=\"symbol\">sE, seu anti-sigma e uma protease, e <font face=\"symbol\">sE não se mostrou auto-regulado, mas regulou o gene do anti-sigma. Análises similares às acima indicaram que o gene pilA, codificando a pilina da fímbria tipo IV, é positivamente regulado por <font face=\"symbol\">sN, enquanto o operon codificando proteínas da fímbria tipo I é regulado negativamente, explicando a maior formação de biofilme e auto-agregação no mutante rpoN. O perfil temporal de expressão da linhagem selvagem J1a12 em carência de nitrogênio foi determinado, além de genes induzidos por carência de nitrogênio via <font face=\"symbol\">sN. Assim, <font face=\"symbol\">sN regula genes de fímbrias e de resposta à carência de nitrogênio em Xylella fastidiosa. / Mutant strains were obtained for the sigma factors <font face=\"symbol\">sE (RpoE) and <font face=\"symbol\">sN (RpoN) in Xylella fastidiosa. The rpoE mutant showed to be sensitive to ethanol and heat shock. Microarray and quantitative RT-PCR analyses and determination of transcription start sites permitted to define the <font face=\"symbol\">sE regulon under heat shock. Co-transcription of the genes encoding <font face=\"symbol\">sE , its anti-sigma factor and a protease was observed, and <font face=\"symbol\">sE did not present auto-regulation, but it regulated the gene encoding the anti-sigma. Similar analyses indicated that the pilA gene, encoding the pilin of the type IV fimbriae, is positively regulated by <font face=\"symbol\">sN, while the operon encoding proteins of the type I fimbriae is negatively regulated, what explains the increased biofilm formation and auto-aggregation in the rpoN strain. Temporal expression profile of wild type strain J1a12 under nitrogen starvation was determined, as well as genes induced by nitrogen starvation via <font face=\"symbol\">sN. Thus, <font face=\"symbol\">sN regulates genes encoding fimbriae and genes for nitrogen starvation response in Xylella fastidiosa.

Les changements climatiques et l'émission de polluants dans l'environnement sont susceptibles d'entrainer des effets croisés sur les communautés et le fonctionnement des écosystèmes. Les changements de température sont notamment susceptibles de modifier le comportement et la toxicité des polluants, et de sensibiliser les organismes aux stress chimiques. Inversement, l'exposition à des polluants peut diminuer la tolérance thermique des espèces ectothermes comme les poissons. Dans ce contexte, nous avons étudié la réponse d'une espèce modèle de poissons en toxicologie aquatique, le Carassin doré (Carassius auratus), soumis à des stress chimique et thermique individuels et combinés. Pour cela, le carassin doré a été exposé à un cocktail d'herbicides et de fongicides à des concentrations réalistes d'un point de vue environnemental à deux températures pendant 96h ou 16 jours. Les réponses ont été observées de l'échelle moléculaire à l'échelle individuelle par des approches omiques (protéomique et métabolomique), biochimiques (cortisol, biomarqueurs de stress oxydant et allocation cellulaire énergétique), indicielles (indices somatiques et de condition) et comportementales (remaniement sédimentaire, activité, exploration et comportement alimentaire). Les résultats montrent que l'exposition des poissons aux stress chimique et thermique individuels entraine une réponse générale de stress impliquant des compensations biochimiques, énergétiques, physiologiques et comportementales. L'absence d'effets sur la santé générale des carassins suggère la mise en place d'une réponse de stress efficace et adaptée, bien que l'hypoactivité observée chez les poissons exposés aux cocktails de pesticides soit susceptible d'entrainer une diminution de leurs performances et de leur fitness. A l'inverse, les carassins exposés aux stress chimique et thermique combinés montrent une inhibition de la réponse générale de stress, avec des effets antagonistes sur la sécrétion de cortisol, l'induction de certains systèmes de défense antioxydants et la compensation énergétique. Des effets plus importants sont en revanche observés au niveau comportemental ainsi qu'une diminution significative de la condition générale des poissons. Ces résultats montrent que l'augmentation de la température sensibilise les poissons à la contamination de l'eau par les pesticides. L'inhibition de la réponse de stress chez des poissons exposés à des mélanges complexes de pesticides dans un écosystème soumis à de multiples contraintes pose de nombreuses questions pour la conservation des espèces dans l'environnement. / Crossed-effects between climate change and chemical pollutions were identified on community structure and ecosystem functioning. Temperature rising affect the toxic properties of pollutants and the sensitiveness of organisms to chemicals stress. Inversely, chemical exposure may decrease the thermal tolerance of ectothermic species, as fish. In this context, we studied the response of a biological model in aquatic toxicology, the goldfish (Carassius auratus), to individual and combined chemical and thermal stresses. In this aim, we exposed the goldfish to environmental relevant concentrations of herbicide and fungicide mixtures at two temperatures for 96 hours or 16 days. The fish responses were assessed from the molecular level to individual endpoints, including omic approaches (proteomic and metabolomic), biochemical analyses (cortisol, antioxidant defenses, cellular energy allocation), indexes (somatic and condition factors) and behavioral assays (sediment reworking, activity, exploration and feeding). Our results showed that individual chemical or thermal stresses induced a general stress response including biochemical, metabolic, physiological and behavioral compensations. The absence of deleterious effect on the global condition of fish suggested the implementation of an efficient and adaptive stress response, while the hypoactivity of fish exposed to pesticide mixtures could entrain a decreased performance and fitness into the wild. At the opposite, the combined chemical and thermal stresses induced reciprocal inactivation of the stress response, with antagonism effect on cortisol secretion, antioxidant defense induction and metabolic compensation. However, increased effect on behavioral traits and decreased global condition of fish were observed. Our study showed that temperature rising sensitized fish to pesticide exposure. Finally, inhibited stress response in fish exposed to pesticide cocktails raises concerns about species conservation an ecosystem under multiple pressures.

X-ray crystallography is one of the most powerful tools for the elucidation of the structure of biological macromolecules such as proteins and viruses. Crystallographic techniques are extensively used for investigations on protein structure, ligand-binding, mechanisms of enzyme catalyzed reactions, protein-protein interactions, role of metal ions in protein structure and function, structure of multi-enzyme complexes and viruses, protein dynamics and for a myriad other problems in structural biology. Crystallographic studies are essential for understanding the intricate details of the mechanism of action of enzymes at molecular level. Understanding the subtle differences between the pathogenic enzymes and host enzymes is necessary for the design of inhibitor molecules that specifically inhibit parasite enzymes. The current thesis deals with the application of biochemical and crystallographic techniques for understanding the structure and function of proteins from two pathogenic organisms – a plant virus Physalis Mottle Virus (PhMV), and a pathogenic bacterium, Salmonella typhimurium and also stress induced proteins from Oryza sativa. The thesis has been divided into seven chapters, with the first four chapters describing the work carried out on PhMV, while the rest of the chapters deal with the studies on stress response proteins from Oryza sativa and Salmonella typhimurium. The first part of the thesis deals with studies on viral capsids. Viruses are obligate parasites that have proteinaceous capsids enclosing the genetic material, which, in the case of small plant viruses, is invariably ss-RNA. X-ray diffraction studies on single crystals of viruses enable visualization of the structures of intact virus particles at near-atomic resolution. These studies provide detailed information regarding the coat protein folding, molecular interactions between protein subunits, flexibility of the N-and C-terminal segments and their probable importance in viral assembly, role of RNA in capsid assembly, nucleic acid (RNA)-protein interactions, the capsid structure and mechanism of assembly and disassembly. The present thesis deals with the capsid structure and analysis of the coat protein (CP) recombinant mutants of PhMV. Virus assembly, one of the important steps in the life cycle of a virus, involves specific interactions between the structural protein and cognate viral genome. This is a complex process that requires precise protein-protein and protein nucleic acid interactions. In fact, most of the biological functional units such as ribosomes and proteosomes also require highly co-ordinated macromolecular interactions for their functional expression. Viruses being simple in their architecture, serve as excellent model systems to understand mechanism of macromolecular assembly and provide necessary information for the development of antiviral therapeutics, especially in animal viruses. PhMV is a plant virus infecting several members of Solanaceae family. It belongs to the tymoviridae group of single stranded RNA viruses. Its genome is encapsidated in a shell comprising of 180 (architecture based on T = 3 icosahedral lattice) chemically identical coat protein (CP) subunits (~ 20,000Da) arranged with icosahedral symmetry. In an earlier phase of work, PhMV purified from infected plant leaves was crystallized in the space group R3 (a = 294.56 Å,  = 59.86). X-ray diffraction data to 3.8 Å resolution were recorded on films by screenless oscillation photography. Using this data of severely limited quality and poor completion (40%), the structure PhMV was determined by molecular replacement using the related turnip yellow mosaic virus (TYMV) structure as the phasing model. There was therefore a need to re-determine and improve the structure, which could be useful for understanding the earlier detailed studies on its biophysical properties. As a continuation of these studies, the present investigations were conceived with the goal of determining the natural top and bottom component capsid structures of PhMV. Investigations were also carried out to examine the possibility of enhancing the diffraction quality of PhMV crystals. The thesis begins with a review of the current literature on the available crystal structures of viruses and their implications for capsid assembly (chapter I). All experimental and computational methods used during the course of investigations are described in chapter II, as most of these are applicable to all the structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. Chapter III describes structural studies on top and bottom components of PhMV. Purified tymoviruses including PhMV are found to contain two classes of particles that sediment at different velocities through sucrose gradients and are called the top (sedimentation coefficient 54 Svedberg units(S)) and the bottom (115S) components. The top component particles are either devoid of RNA or contain only a small subgenomic RNA (5%) while the bottom component particles contain the full length genomic RNA. Only the bottom component is infectious. The top and bottom components were separately crystallized in P1 and R3 space groups, respectively. It is of interest to note that crystals of the bottom component obtained earlier belonged to R3 space group while recombinant capsids that lack of full length RNA as in natural top component crystallized in the P1 space group. A polyalanine model of the homologous TYMV was used as the phasing model to determine the structures of these particles by molecular replacement using the program AMoRe. The refinement of top and bottom component capsid structures were carried out using CNS version 1.1 and the polypeptide models were built into the final electron-density map using the interactive graphics program O. The quality of the map was sufficient for building the model and unambiguous positioning of the side chains. There is a significant difference in the radius of the top and bottom component capsids, the top component being 5 Å larger in radius. Thus, RNA makes the capsid more compact, even though RNA is not a pre-requisite for capsid assembly. Partially ordered RNA was observed in the bottom component. The refined models could form the basis for understanding the architecture, protein-protein interactions, protein-nucleic acid interactions, stability and assembly of PhMV. Chapter IV provides a detailed description of the mutations carried out on PhMV coat protein towards enhancing the diffraction quality of crystals. The gene coding for PhMV coat protein (PhMVCP) and several of its deletion and substitution mutants were originally cloned in pRSETC and pET-21 vectors by Mira Sastri and Uma Shankar in Prof. Savithri’s laboratory at the Department of Biochemistry. It was observed that the recombinant intact coat protein and several mutants lacking up to 30 amino acids from the N-terminal end could assemble into empty shells resembling the natural top component. None of these deletion mutants crystallized in forms that diffracted to high resolution. Based on the intersubunit contacts observed, three more site-specific mutants were designed. These three mutants were expressed in BL21 (DE3), purified and crystallized. Even these mutant crystals did not diffract to high resolution. The polypeptide fold of PhMV coat protein therefore was carefully examined for probable reasons. It was found that PhMV subunit has three major cavities. Three cavities are likely to increase the flexibility of protein subunits, which in turn may result in crystals of poor quality. Mutations V52W, S158Q and A160L were shown to fill up these cavities and with the view of obtaining better crystals. These site specific mutations were carried out the mutant proteins were purified. It was shown that the recombinant capsids are stable and possess T=3 architecture. Two mutants were crystallized and a data set for V52W extending to 6.0 Å resolution could be collected. Due to the limited resolution, further work was not pursued. It is plausible that the triple mutant will diffract to higher resolution. The second part of the thesis deals with stress response proteins from Oryza sativa and Salmonella typhimurium. It is known that viral infection and abiotic and biotic stresses induce a network of proteins in plants. Chapter V presents a review of the current literature on stress proteins, focusing mainly on Oryza sativa and S. typhimurium stress response proteins. Chapter VI describes the over expression of stress proteins SAP1 and SAP2 from rice. These stress related proteins confer tolerance to cold, dehydration and salt stress in rice. These proteins have been cloned in the expression vector pEt-28(a) and expressed in E. coli strain BL21 CodonPlus(DE3)RIL. The proteins were purified and crystallization trials were made. However, there were no hits. In an attempt to get crystals, nine deletion constructs of SAP1 were designed eliminating potentially disordered and unfolded regions based on a bioinformatics analysis. Crystallization trails are being carried out on three of the constructs. Structural studies on a universal stress protein from Salmonella typhimurium, which shares homology with the rice universal stress proteins, was initiated. Apart from this, several other stress related proteins of Salmonella typhimurium have also been selected for structural and functional studies. These include YdaA, YbdQ, Yic, Ynaf, Yec, Spy and Usb. All these were cloned and expressed in E. coli. Out of seven proteins, Ynaf, YdaA and YbdQ were found in the soluble fraction and were expressed in quantities suitable for structural studies. I could crystallize YdaA and Ynaf. X-ray diffraction data to resolutions of 3.6 Å and 2.3 Å were collected on crystals of YdaA and YnaF, respectively. A tentative structure of YnaF has been obtained. Further attempts to determine these structures are in progress. Biophysical, Biochemical functional characterization of YdaA and YnaF proteins are described. Structural studies on mannose-6-phosphate isomerase, an enzyme related to stress regulatory proteins from S. typhimurium are dealt with in Chapter VII. Mannose 6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The structure could be solved in its apo and holo forms (with two different metal atoms, Y3+ and Zn2+), and complexed with the cyclic form of the substrate fructose 6-phosphate (F6P) and Zn2+. Isomerization involves acid/base catalysis with proton transfer between C1 and C2 atoms of the substrate. Lys 132, His 131, His 99 and Asp 270 are close to the substrate and are likely to be the residues involved in proton transfer. Interactions observed at the active site suggest that the ring opening step is catalyzed by His 99 and Asp 270. An active site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. The metal ion is not close to the substrate atoms involved in proton transfer. Binding of the metal induces structural order in the loop consisting of residues 50-54. Hence, the metal atom does not appear to play a direct role in catalysis, but is probably important for maintaining the architecture of the active site. Based on these structures and earlier biochemical work, a probable isomerization mechanism has been proposed. The thesis concludes with a brief discussion on the future prospects of the work. The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis:

The increasing market demand for fresh produce promotes a keen interest in developing a rapid, sensitive and reliable method for monitoring plant health and determining the shelf-life of postharvest produce. The objective of this study is to explore the capability of Surface-enhanced Raman spectroscopy (SERS) in these applications. SERS integrates Raman spectroscopy which measures molecular vibrations and nanotechnology which enhances the weak Raman signals. Herein, we developed two SERS methods based on a surface detection approach using nanoparticles solution and a headspace detection approach using gold nanoparticles (AuNPs) fibers, to detect biochemical changes during postharvest storage of arugula leaves. Compared with surface detection, the headspace detection revealed significant spectral changes during the storage, particularly in the shifts around 500, 950 and 1030 cm-1. These changes analyzed using principal component analysis (PCA) to establish a prediction model for shelf-life determination. Through analyzing reference standard compounds, we identified the dimethyl disulfide (DMDS), 1-propanethiol and methanethiol (MT) were most likely to account for the signature spectra of headspace arugula at the late storage period due to the activities of spoilage bacteria. The headspace detection method was also applied to monitor the stress responses of living basil to abiotic stresses (pesticide/salinity). However, the volatile analysis of the basil plants response to abiotic stresses (pesticide/salinity) showed indistinctive results. In conclusion, the headspace detection based on SERS provides a new strategy for quality monitoring of fresh produce in the food industry.

Kvasinky žijú v neustále sa meniacom prostredí. Aby prežili tieto výkyvy ich okolitého prostredia, musia sa vedieť rýchlo a efektívne prispôsobiť novým podmienkam. Jedným z aspektov takejto bunkovej adaptácie je reorganizácia génovej expresie na program vyžadovaný pre rast v novom prostredí. Dôsledkom tejto reorganizácie genómu sú zmeny v metabolizme a fyziológií kvasiniek. Molekulárna odpoveď bunky určuje či sa organizmus adaptuje, prežije alebo zahynie. Predložená dizertačná práca sa zaoberá štúdiom vplyvu environmentálnych zmien na genóm a metabolóm vybraných karotenogénnych kvasiniek. Kvasinky boli kultivované jednak za optimálnych podmienok a jednak v oxidačnom a ozmotickom strese a na rôznych odpadových materiáloch (srvátke, zemiakovom odpade a pod.). V prítomnosti stresu bola pozorovaná zvýšená produkcia biologicky významných karotenoidov. Takáto obohatená biomasa môže nájsť svoje uplatnenie v biotechnologickom priemysle, napr. ako krmivo pre zvieratá. Možnosťou štúdia odozvy mikroorganizmov na environmentálny stres je aj príprava transformantov s deléciou vybraných génov a ich analýza. V ďalšej časti práce bola prevedená delécia vybraných génov kvasinky Schizosaccharomyces pombe. Zvolená technika je založená na knockoute konštruktov, ktoré obsahujú regióny homologické s deletovaným génom. Analýzou vytvorených transformantov boli identifikované proteíny potrebné pri meiotickej segregácií chromozómov.

ROLE OF THE YXKO GENE OF BACILLUS SUBTILIS IN RESPONCE TO ENVIRONMENTAL STRESS Abstract Mutation of the yxkO gene, which encodes a putative ribokinase and belongs to the σB general stress response regulon, leads to reduced salt tolerance under potassium limitation in Bacillus subtilis. The biological function of the yxkO gene has not been determined yet, but it may be involved in the high affinity potassium uptake system, which has been described in Escherichia coli in contrast to Bacillus subtilis. Our goal was to describe another features of a mutant in the yxkO gene and to try to propose the role of this gene. Using the integration vector pMutin4, we prepared a Bacillus subtilis strain MP2 with a yxkO gene inactivation. The MP2 strain displays limited growth in a rich medium and it is a sensitive strain to tetracycline. Furthermore, this strain is unable to form endospores and the cells are longer, which indicates a septum formation defect. We accomplished a 2-D protein gel analysis to compare expression profiles of the MP2 strain and the 1A680 standard strain after salt and ethanol stress. The MP2 strain shows changes in productions of some energy metabolism enzymes and flagellin protein. We conclude that yxkO is a regulatory gene, whose product has a pleiotropic effect on many of cell functions.

Plants secrete peptide ligands and use receptor signaling to respond to stress and control development. Understanding the signaling mechanisms and associated molecular trafficking is key to improving plant health and productivity for food, fiber and energy applications. However, one of the challenges to elucidating communication pathways in plants is to study the trafficking of molecules and signals iteratively and non-destructively. This study focuses on using fiber-optic fluorescence microscopy to image live plants iteratively and non-destructively after delivering both labeled and unlabeled phytosulfokine (PSK) into the plant. PSK is a sulfated peptide hormone involved in the regulation of plant cell division and growth via specific receptors, PSKRs. It also plays a role in regulating how plants are able to tolerate stress conditions. The microscope provides two-color (FITC/TRITC) optics and provides high-resolution (3–5 µm) epifluorescence micrographs via a 1-m coherent imaging fiber and a GRIN objective lens. To obtain high-quality images, the fiber was mounted either to a conventional upright microscope body equipped with a leaf compressor, or to a leaf clip with 5-axis positioning (X–Y–Z plus pitch and yaw) mounted on an extensible arm. PSK and TAMRA-labelled PSK were delivered into the roots of various Arabidopsis thaliana genotypes (wt; receptor-deficient: pskr1/pskr2; and tagged receptor overproducing: PSKR1‑GFP), and their movement in roots and leaves was tracked with the fiber-optic fluorescence microscope. Peptide trafficking was successfully observed in live plants non- destructively, confirming that PSK is mobile in both wt and receptor-deficient plants. Preliminary results suggest that the level of receptor PSKR1 may change in response to PSK, and that levels of PSKR1, PSKR2 or both may impact the trafficking of PSK. Understanding how PSK is trafficked in plants will offer insights into how we can improve plants health and productivity.