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"The in vitro addition of plant growth regulators (i.e. phytohormones) to Agrobacterium transformed hairy root cultures affects morphological and biochemical changes, resulting in altered growth and secondary metabolite accumulation rates in root tissues. Significant increases in both growth and secondary product accumulation have been observed, upon incubation with phytohormones, in some species. Consequently, the use of phytohormones in vitro has received increasing attention as a potential means for increasing those plant secondary products notoriously produced in small quantities. However, currently little is known about the specific effects of phytohormones on growth and secondary metabolism. The Chinese herb Artemisia annua L. produces artemisinin, an effective antimalarial therapeutic. Efforts to increase the amount of artemisinin via chemical synthesis or field-grown crops have met with huge costs and disappointingly low yields, respectively. Agrobacterium transformed hairy root cultures of A. annua (Clone YUT16) produce artemisinin and undergo rapid growth compared to non-transformed, making them a good model system to study secondary metabolite production. Demonstrated herein is the first definitive evidence, by any hairy root species, of a favorable response to exogenous combinatorial hormone application as well as the development of a two-stage culture system alluding to optimal growth and artemisinin production conditions in A. annua hairy roots. Furthermore, analysis of artemisinin and biomass accumulation in A. annua hairy roots in the presence of phytohormones has revealed effective individual as well as combinatorial phytohormone concentrations suitable for increasing single and bulk root growth, and artemisinin production. The effectiveness of an optimal phytohormone combination, with respect to time of addition, its relationship to inoculum size, and its combination with the provision of fresh nutrients and or mechanical stress to the roots is also described resulting in artemisinin yields of up to 0.8 ìg/g F.W. Although the findings contained herein are not yet optimized they do, however, argue for the potential usefulness of a two-stage production scheme using phytohormones to increase plant secondary metabolite production in vitro."

Carotenoids are precursors of hormones and signaling molecules across all kingdoms of life. An increasing body of evidence suggests the presence of yet unidentified carotenoid-derived metabolites (apocarotenoids) with developmental and regulatory functions, besides the known plant hormones abscisic acid (ABA) and strigolactones (SLs). Generally, apocarotenoid synthesis is initiated by carotenoid cleavage dioxygenases (CCDs), which constitute a ubiquitous family of non-heme iron enzymes. In SL biosynthesis, an iron-binding cis/trans-isomerase, DWARF 27 (D27) converts all-trans-β-carotene into 9-cis-β-carotene. This reaction is followed by a double bond cleavage at 9, 10 position, mediated by the stereospecific CCD7. The cis-configured cleavage product of CCD7, 9-cis-β-apo-10’-carotenal, is simultaneously cleaved, triple-oxygenated and rearranged by CCD8, to produce carlactone (CL). CL is a central metabolite and the precursor of a wide range of SLs. The aim of this work is to investigate whether CCD8 synthesize CL-like compounds from other 9-cis-configured apocarotenoids to confirm their presence and synthesis in planta. We showed that CCD8 enzymes from different plants produce a hydroxylated carlactone (3-H-CL) from 9-cis-3-OH-β-apo-10’-carotenal in vitro. In addition, we detected 3-H-CL in Nicotiana benthamiana leaves transiently expressing the CL biosynthesis enzymes from rice and Arabidopsis. 3-H-CL is biologically active, as shown by Striga hermonthica seed germination assay and by its effect on the high-tillering phenotype of the rice d10 mutant. We also confirmed that 3-H-CL is a natural metabolite by detecting it in roots of the rice SL perception mutant d14. In a second project, we investigated the activity of three rice CCDs in vitro and showed that one of them (zaxinone synthase; ZAS) is an apocarotenoid cleavage enzyme with a clear preference for the substrate all-trans-3-OH-β-apo-10’-carotenal, as suggested by a kinetic study. ZAS produces two products, the C18 ketone zaxinone and an unstable C9 dialdehyde that could be identified by LC-MS after derivatization. Activity tests were performed with crude lysates of overexpressing Escherichia coli cells and with purified enzyme. We established that zaxinone is a natural metabolite present in planta. Investigations of a corresponding rice mutant (zas) and activity bioassays performed by our group demonstrate that zaxinone a novel signaling molecule required for normal rice growth and development.

Les modèles climatiques indiquent qu’il est très probable que les végétaux soient de plus en plus exposés à deux facteurs de stress environnementaux : l’ozone troposphérique (O3) et le déficit hydrique du sol, tous deux pouvant provoquer un stress oxydant pour le végétal. Dans des conditions naturelles, ces deux facteurs peuvent être concomitants ou se succéder. L’impact de l’O3 et de la sécheresse nécessite donc une attention particulière. Afin de déterminer les réponses de défense mises en place par les arbres, deux génotypes de Populus nigra x deltoides (Carpaccio et Robusta) ont été exposés aux contraintes séparées ou à leur combinaison en conditions contrôlées dans des chambres de culture. Pour explorer les effets des stress et l’interaction entre les deux contraintes, nous avons ciblé les deux premiers niveaux de défense des plantes que sont le contrôle de l’ouverture/fermeture des stomates et les processus de détoxication cellulaire. Nos résultats montrent que Carpaccio et Robusta sont tous deux relativement tolérants à une sécheresse modérée grâce à un contrôle efficient des stomates. Face à l’O3, cependant, les deux génotypes adoptent des stratégies de réponse différentes : un évitement important pour Carpaccio et une maximisation de l’assimilation au détriment des feuilles pour Robusta. Cela se traduit par une différence de fermeture des stomates. Les deux génotypes ne font alors pas face au même flux d’O3 entrant dans les feuilles, ce qui impacte la détoxication cellulaire, dans laquelle le glutathion semble jouer un rôle majeur. En lien avec les modifications de capacité antioxydante, l’activité des enzymes du cycle ascorbate-glutathion (MDHAR, DHAR et GR) et/ou l’expression des gènes codant pour ces protéines sont modifiées. En combinaison de stress, le déficit hydrique protège le végétal du stress oxydant induit par l’O3 en amplifiant la fermeture des stomates. En revanche, la croissance de l’arbre est impactée par l’effet additif des deux contraintes. De plus, l’induction de voies de régulation hormonales différentes par les deux contraintes pourrait modifier le « cross-talk » complexe régulant la réponse au stress combiné. Enfin, dans le cas d’une succession de stress, l’exposition à l’O3 avant un épisode de sécheresse impacte faiblement la réponse de l’arbre. Cependant, un ralentissement de la fermeture des stomates induit par l’O3 est observé malgré l’arrêt de la fumigation. Il est donc nécessaire de prendre en compte le ralentissement et la fermeture des stomates induit par l’O3 et le déficit hydrique dans les modèles de conductance stomatique utilisés pour calculer l’indicateur du flux d’O3 entrant, le PODy (Phytotoxic Ozone Dose above a threshold of y nmol O3 m-2.s-1). / Climate models indicate that it is very likely that plants will be more and more exposed to two environmental stressors: ground-level ozone (O3) and soil water deficit, both causing oxidative stress to the plant. Under natural conditions, these two factors can be concomitant or successive. Therefore, the impact of O3 and drought requires special attention. In order to determine the defensive responses adopted by trees, two genotypes of Populus nigra x deltoides (Carpaccio and Robusta) were exposed to separate or combined stresses under controlled conditions in growing chambers. To explore the effects of stresses and their interaction, we targeted the plant’s first two levels of defence: i) the control of stomatal opening and closing, ii) the cellular detoxification processes. Our results show that both Carpaccio and Robusta are relatively tolerant to moderate drought thanks to an efficient stomatal control. However, different response strategies were adopted by the two genotypes to cope with O3. For Carpaccio, the strategy is avoidance, and for Robusta, the strategy is maximization of net CO2 assimilation at the expense of leaves. This results in a difference in the stomatal closure. The two genotypes do not face the same flow of O3 entering the leaves. This impacts cellular detoxification in which glutathione seems to play a major role. Also, the activity of ascorbate-glutathione cycle enzymes (MDHAR, DHAR and GR) and/or the expression of genes encoding these proteins are modified. Under combined stresses, the water deficit protects the plant from the O3-induced oxidative stress by amplifying the stomatal closure. Nevertheless, the tree growth is impacted by the additive effect of the two stresses. Furthermore, the induction of different hormonal regulatory pathways by the two stressors could modify the complex "cross-talk" regulating the response to combined stress. Finally, in the case of a succession of stresses, exposure to O3 prior to a drought episode has a weak impact on the tree's response. However, O3 induced a stomatal sluggishness in closure despite the cessation of fumigation. It is therefore necessary to take into account stomatal closure and sluggishness induced by O3 and water deficit in the stomatal conductance models used to calculate the indicator of O3 flux inside the leaves, PODy (Phytotoxic Ozone Dose above a threshold of y nmol O3 m-2.s-1).

Les plantes ont la capacité à former de nouveaux organes grâce à une croissance continue assurée par une réserve de cellules souches au sein de structures spécifiques, les méristèmes. Les méristèmes floraux diffèrent des méristèmes végétatifs par leur caractère déterminé aboutissant à la production des fleurs. Le gène IMA (INHIBITOR OF MERISTEM ACTIVITY) code une protéine contenant un motif «doigt à zinc» (MIF) régulant les processus développementaux de la fleur et des ovules chez la tomate. En effet, IMA inhibe la prolifération cellulaire au cours de la terminaison florale en agissant sur l’expression du gène WUSCHEL, responsable du maintien du pool de cellules souches et contrôle le nombre de carpelles (Sicard et al., 2008). De plus, les protéines IMA et son orthologue chez Arabidopsis, MIF2, modulent la réponse à certaines phytohormones. De manière identique à la protéine MIF1 (Hu and Ma, 2006), IMA/MIF2 régule négativement la réponse aux brassinostéroïdes, à l’auxine, aux cytokinines et aux gibbérellines mais positivement la réponse à l’acide abscissique suggérant une fonction commune des protéines MIF dans les voies de réponse aux phytohormones. Un criblage d’une banque d’ADNc par la technique de double hybride a permis de révéler l’interaction entre les protéines IMA/MIF2 et une sous-unité du complexe signalosome, CSN5. De façon intéressante, les plantes mutantes csn5 d’Arabidopsis montrent de nombreuses altérations phénotypiques telles qu’un aspect buissonnant résultant de la perte de la dominance apicale, et une altération de la réponse à l’obscurité et à l’auxine. Ces phénotypes sont fortement ressemblants aux phénotypes des plantes MIF1OE d’Arabidopsis (Hu and Ma, 2006) et des plantes IMAOE de tomate (Sicard et al., 2008). Les résultats obtenus au cours de ce projet montrent que la protéine IMA inhibe la fonction du complexe signalosome grâce à son interaction avec la protéine CSN5. / Plants have the ability to form new organs as a result of indeterminate growth ensured by specific regions of pluripotent cells, called meristems. Flowers are produced by the activity of floral meristems which differ from vegetative meristems in their determinate fate. The INHIBITOR OF MERISTEM ACTIVITY (IMA) gene encoding a Mini Zinc Finger (MIF) protein from tomato (Solanum lycopersicum) regulates the processes of flower and ovule development. IMA inhibits cell proliferation during floral termination, controls the number of carpels during floral development and acts as a repressor of the meristem organizing centre gene WUSCHEL (Sicard et al., 2008). We demonstrated that IMA and its Arabidopsis ortholog MIF2 is also involved in a multiple hormonal signalling pathway, as a putative conserved feature for plant MIF proteins (Hu and Ma, 2006). Alike Arabidopsis MIF1, IMA/MIF2 regulates negatively BR, auxin, cytokinin and gibberellin signalling and positively ABA signaling. Using yeast two-hybrid screening experiments, we identified a strong protein-protein interaction between IMA and the signalosome subunit 5 (CSN5). Interestingly the csn5 mutant in Arabidopsis displays pleiotropic developmental defects such as a bushy phenotype originating from the loss of apical dominance and the alteration in sensitivity to darkness and auxin signals. These phenotypes are strikingly similar to what was described for Arabidopsis MIF1 (Hu and Ma, 2006) and tomato IMA overexpressors plants (Sicard et al., 2008), respectively. Taken together our data strongly suggest that IMA may act as an inhibitor of CSN function through its physical interaction with SlCSN5. The observed converse effects of IMA/MIF2 overexpression or deregulation on plant development and the abundance of developmental marker genes further support the notion of a CSN inhibitory control, since the COP9 signalosome through the specific deneddylation activity of the CSN5 subunit regulates plant hormone signalling.

The present investigation was undertaken as a preliminary study to learn the response of Escherichia coli cells grown under identical experimental conditions to various concentrations of indoleacetic acid, gibberellic acid, and kinetin alone, and in combination with one another.

Darbo objektu pasirinkti Petunia genties augalai, siekiant įvertinti, šių plačiai naudojamų balkonų ir lauko želdinimui gėlių, fitohormonų ir šviesos – tamsos įtaką audinių regeneracijai. Atskirų veiksnių poveikiai literatūroje dažnai aprašomi, tačiau kompleksinis veikimas analizuojamas mažai. Tokio tipo tyrimai yra svarbūs tiek mokslui, tiek verslui. Atlikto tyrimo tikslas ištirti kompleksinę fitohormonų ir šviesos įtaką Petunia regeneracijai in vitro. Tikslui pasiekti atlikta mokslinės ir metodinės literatūros, susijusios su darbo tema, analizė. Atlikti eksperimentai su izoliuotais Petunia eksplantais, kultivuojant juos skirtingos sudėties terpėse šviesoje ir tamsoje bei įvertinta eksplantuose įvykusi regeneracija. Pagrindinis metodas tyrimams atlikti – 1IAR ir 5BAP fitohormonų integravimas į MS terpę. Metodo akcentas – šviesos ir tamsos sąlygos, kuriose buvo kultivuojami eksplantai terpėje su fitohormonais ir kontrolinėje (be fitohormonų) MS terpėje. Atlikus tyrimą nustatyta, kad P. nana compacta stiebo ir lapo eksplantai pasižymi 100% regeneracija kultivuojami terpėje su fitohormonais tamsoje. Nustatyta, kad lapų eksplantuose rizogenezė indukuojama beveik 2 kartus rečiau nei iš stiebų izoliuotuose audiniuose. Rezultatų analizė parodė, kad kultivuojant stiebo dalis, 56% daugiau ūglių eksplantai suformuoja terpėje su fitohormonais ir visais atvejais tamsoje jų buvo mažiau nei šviesoje. Daugiausiai, 3,1 ūglių/eksplante, buvo P. grandiflora MS (1IAR:5:BAP) regenerantuose... [toliau žr. visą tekstą] / Plants of Petunia genus, flowers widely used for planting on balconies and outdoors, have been selected as the object of the Paper in order to estimate the influence of phytohormones and light – darkness on regeneration of tissues. The influence of particular factors is often described in literature, however, complex influence thereof is analysed scarcely. Researches of this type are very important for both science and business. Aim of the research carried out is to study the complex influence of phytohormones and light on regeneration in vitro of Petunia. Analysis of scientific and methodical readings related to subject of the Paper has bee carried out in order to achieve the above aim. Experiments with isolated Petunia explants have been made by cultivating them in media of different compositions in light and darkness and regeneration that took place in the explants was evaluated. The key research method was integration of 1IAA and 5BAP phytohormones into MS medium. Focus of the method – conditions of light and darkness under which the explants were cultivated in the medium with phytohormones and control (without phytohormones) MS medium. Upon completion it was determined 100% P. nana compacta footstalk and leaf explants regeneration cultivating them in the medium with phytohormones in darkness. It has been determined that rhizogenesis induced in leaf explants was above 2 times less than in case of footstalk in isolated tissues. The results analysis showed that, in case... [to full text]

The literature on the effects of phytohormone on algae is clouded with contradictory reports. Reports have been published which substantiate and deny the effects of phytohormones in enhancing the growth and developmental processes in algae. The overall aim of this study was to investigate the response, if any, of the phytohormones indole-3-acetic acid (IAA), gibberellic acid A3 (GA) and kinetin on the physiology of the green alga, Scenedesmus quadricauda. Results obtained for the uptake of 14^C-IAA an(j l4C-kinetin by Scenedesmus strongly support the presumption that the alga does not absorb the hormones. The retention of the phytohormones by the alga is due to adsorption, and is independent of hormone concentration. Most of the label was adsorbed by the outer pectic layers of the cell wall.

Apoplastic barriers (exodermis and endodermis) control free movement of substances by apoplast, which is achieved by specific cell wall modifications. Differentiation of these barriers is to some extent variable. It has been demonstrated many times that the differentiation of root apoplastic barriers is strongly influenced by unfavourable environmental conditions. Many stress factors accelerate the deposition of apoplastic barriers. This work deals with the relationship between availability of nutrients and development of root barriers and mechanisms of coordination of developmental processes in roots, including the involvement of auxin and other phytohormones in the coordination of these processes. The exodermis developmental plasticity is followed in maize (Zea mays L.) under conditions of various nutrient deficiencies. The most important results are presented by anatomical analysis, which shows a significant acceleration of exodermis and endodermis differentiation in the N and P deficiency on the contrary, the slowing of differentiation in the absence of Fe and K. This effect is apparently a result of systemic rather than local root system responses because it was not observed with localized deficiency. This thesis also test the influence of some phytohormones on the development of apoplastic...

The debilitating impact of climate change on crop production remains an issue of global concern. Climate change has over the years been a driving force of extreme weather conditions that can adversely impact the growth, development, and yield of crops. Plants are able to combat the effects of stress conditions using complicated signaling networks and pathways. Several chemicals and phytohormones have been identified as part of these signaling pathways. However, the role of phytohormones in facilitating the transmission of extracellular stress stimuli into intracellular responses in plants remains unclear. This challenge is a consequence of the difficulty in studying real-time plant trafficking mechanisms in live plants and understanding how phytohormones facilitate this process. The objective of this study is to explore the mechanism of phytosulfokine trafficking in Arabidopsis thaliana with the aid of a fiber-optic fluoresce microscope that was built specifically for this purpose. Phytosulfokine (PSK) is a peptide hormone that is involved in both plant growth and stress response signaling. PSK and tetramethyl-rhodamine labelled PSK (TAMRA-PSK) were delivered to the roots and leaves of different Arabidopsis thaliana genotypes (wild type, PSK receptor deficient, overly expressed PSK receptor). With the aid fluorescence micrographs captured by the fiber-optic microscope PSK was observed to be transported form the abaxial surface of the leaf to the adaxial surface, the movement of PSK from the roots to the leaves was also observed with significant difference in mobility in the different plant genotypes. The more effective phytosulfokine mobility observed in the genotype with overly expressed PSK receptors suggests that PSK mobility is receptor dependent. Further quantitative analysis via different extraction methods and HPLC analysis will test the amount free PSK and receptor bound PSK in root and leaf tissues to better understand the mechanism of phytosulfokine in plant signaling.