Our ability to control malaria has been challenged by increasing antimalarial resistance. Plasmodium falciparum undergoes dormancy in the blood stages which is hypothesized to be a means by which they are able to survive under drug pressure. This helps select for resistant parasites which grow following removal of drug. The mechanisms behind dormancy and the subsequent recrudescence are not fully understood but translating knowledge from related organisms which undergo a similar phenomenon might shed some light. Higher plants utilize dormancy during the early development stages to survive under unfavorable conditions, increasing fitness of the seedling and ensuring viability when this is released and it develops into a mature plant. Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate this in response to environmental cues. We have found that both can be supplemented to dihydroartemesinin-induced dormant parasites to stimulate early recovery. Fluridone, an ABA inhibitor that releases dormancy in plants, was found to prolong it and cause a delay in recrudescence. These effects were observed in artemisinin sensitive and resistant strains. The apicoplast is required for recovery and supplementation of essential isoprenoid, isopentyl pyrophosphate (IPP), in apicoplast deficient parasites is sufficient enough to compensate for the lack of the organelle in antibiotic treated parasites. IPP plays an important role in development and metabolism of blood stage parasites as a key component of numerous secondary metabolites and protein activity by prenylation of isoprenoids. Its role in dormancy has not been explored prior to this study. Carotenoids are long-chained ABA precursors consisting of two molecules of geranylgeranyl pyrophosphate (GGPP). Several carotenoids as well as enzymes in that pathway have been identified in the blood stages of P. falciparum. The Apicomplexan parasite, Toxoplasma gondii synthesizes ABA, where it plays a role in signaling and development. To date ABA has not been detected in P. falciparum due to limitations in methods previously utilized. We have found that parasites with fosmidomycin inhibition of isoprenoids can be rescued with GGPP supplementation which we planned to use to further elucidate the carotenoid biosynthetic pathway. We hypothesized that Plasmodium has retained the ability to biosynthesize ABA and aimed to confirm this. We developed a novel method to label GGPP with 13C on three of its isoprene units. This would be used to metabolically label isoprenoid inhibited P. falciparum for incorporation through the carotenoid pathway for detection of 13C-ABA.

Philosophiae Doctor - PhD / Global climate change has resulted in altered rainfall patterns, causing annual losses in maize crop yield due to water deficit stress. Therefore, it is important to produce maize cultivars which are more drought-tolerant. This not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of water deficit stress. The RD22-like protein subfamily is expressed in response to water deficit stress. Members of the RD22-like subfamily include AtRD22, GmRd22 and BnBDC1 which have been identified in Arabidopsis thaliana, Glycine max and Brassica napus respectively. This study aims at characterising two putative maize RD22-like proteins (designated ZmRd22A and ZmRd22B) by identifying sequence/domain features shared with characterised RD22-like proteins. Semi-quantitative and quantitative PCR techniques were used to examine the spatial and temporal expression patterns of the two putative maize Rd22-like proteins in response to, water deficit stress and exogenously applied abscisic acid in the roots and 2nd youngest leaves of maize seedlings. Using an in silico approach, sequence homology of the two putative maize Rd22- like proteins with AtRD22, GmRD22 and BnBDC1 has been analysed. Online bioinformatic tools were used to compare the characteristics of these Rd22-like proteins with those of the two maize proteins. It was shown that the putative maize RD22-like proteins share domain organisation with the characterised proteins, these common features include a N-terminal hydrophobic signal peptide, followed by a region with a conserved amino acid sequence, a region containing several TxV (x is any amino acid) repeat units and a C-terminal BURP domain-containing the conserved X₅-CH-X₁₀-CH-X₂₃-₂₇-CH-X₂₃-₂₆-CH-X₈-W motif. The putative maize Rd22-like protein appears to be localized in the apoplast, similarly to AtRD22, GmRD22 and BnBDC1. Analysis of the gene's promotor regions reveals cis-acting elements suggestive of induction of gene expression by water deficit stress and abscisic acid (ABA). Semi-quantitative and quantitative real time PCR analysis of the putative maize RD22-like gene revealed that the genes are not expressed in the roots. Exposure to water deficit stress resulted in an increase of ZmRD22A transcript accumulation in the 2nd youngest leaves of maize seedlings. ZmRD22A was shown to be non-responsive to exogenous ABA application. ZmRD22B was highly responsive to exogenous ABA application and responded to water deficit stress to a lesser degree. Transcript accumulation studies in three regions of the 2nd youngest leaves in response to water deficit stress showed that ZmRd22A transcripts accumulate mainly at the base and tips of the leaves. A restricted increase in ZmRD22A transcript accumulation in the middle of the leaves was observed. ZmRD22B showed a similar, but weaker transcript accumulation pattern in response to water deficit stress. However, ZmRD22B showed increased transcript accumulation in the middle region of the leaves. In response to exogenous ABA application, ZmRd22B exhibited high transcript accumulation at the base of the 2nd youngest leaves, with the middle showing higher transcript accumulation than the tip of the leaves. It was concluded that ZmRD22A and ZmRD22B share the domain organisation of characterised RD22-like proteins as well as being responsive to water deficit stress, although only ZmRD22B was shown to be responsive to exogenous ABA application. / National Research Foundation (NRF)

N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. The abi3-1 mutant showed no expression of ABI3 and AtHVA22B, but RD29B expression remained similar to wildtype seedlings, suggesting an alternate mechanism for NAE and ABA interaction. Taken together, these data suggest that NAE metabolism acts through ABI3-dependent and independent pathways in the negative regulation of seedling development.

La sequía y la salinidad representan estreses ambientales que afectan de forma crítica el crecimiento de las plantas y limitan enormemente su potencial agrícola. La fitohormona ácido abcísico (ABA) juega un papel fundamental en la coordinación de la respuesta y adaptación de las plantas a este tipo de estreses, así como en la regulación del crecimiento y desarrollo vegetal. Elementos intermediarios de la ruta de señalización ya habían sido caracterizados, pero aún se desconocía el mecanismo de percepción y transducción de señal de la hormona. Este trabajo de tesis ha contribuido a la caracterización de una nueva familia de receptores intracelulares de la hormona ABA, formada por 14 miembros y denominada PYR/PYL (de pyrabactin resistance / PYR1-like) /RCAR (de Regulatory Component of Abscisic acid Receptor), y a su caracterización estructural y bioquímica. Estas proteínas son capaces de unir de forma específica la hormona ABA. La unión de la hormona induce en estos receptores un cambio conformacional, que les permite regular la actividad de los reguladores negativos de la ruta: fosfatasas del grupo A como ABI1, ABI2 o HAB1 ( Leung et al., 1994; Meyer et al.,1994; Saez et al., 2004). Para la caracterización de estos receptores se han llevado a cabo abordajes genéticos, bioquímicos, de calorimetría y estudios estructurales. / Santiago Cuéllar, J. (2011). Structural insights into ABA perception and signalling: structure of ABA receptor PYR1 [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/13260 / Palancia

Seeds sown in the fall to restore sagebrush (Artemisia spp.) steppe plant communities could experience high mortality when they germinate and seedlings freeze during the winter. Delaying germination until the risk of frost is past could increase seedling survival. We evaluated the use of abscisic acid (ABA) to delay germination of Elymus elymoides, Pseudoroegneria spicata, and Linum perenne. The following treatments were applied: uncoated seed, seed coated with ABA at 2.2, 4.4, 8.8, 13.2, and 17.6 g of active ingredient kg-1 of seed, and seed coated with no ABA. The influence of seed treatments on germination were tested at five different incubation temperatures (5-25°C). The lowest application rate of ABA had no significant influence on germination percentage but higher application rates showed a decline. All concentrations of ABA tested delayed germination, especially at low incubation temperatures. For example, the time required for 50% of the seeds to germinate at 5°C was increased with the use of the lowest ABA application rate by 56, 61, and 14 days, for E. elymoides, P. spicata, and L. perenne, respectively. Quadratic thermal accumulation regression models were developed for each species and treatment to predict progress toward germination. For the two grasses, models had sufficient accuracy (R2 = 0.61- 0.97) to predict germination timing using field seedbed temperatures. Equations for L. perenne were less accurate (R2 = 0.03-0.70). Use of these models will allow testing whether ABA will delay germination sufficiently to avoid winter frost periods and provide the basis for future field tests.

No description available.

Indoor horticulture offers a promising solution for sustainable food production and is becoming increasingly widespread. However, it incurs high energy and cost due to the use of artificial lighting such as high-pressure sodium lamps, fluorescent light or increasingly, the light-emitting diodes (LEDs). The energy efficiency and light quality of currently available lighting is suboptimal, therefore less than ideal for sustainable and cost-effective large-scale plant production. Here, we demonstrate the use of high-powered single-wavelength lasers for indoor horticulture. Lasers are highly energy-efficient and can be remotely guided to the site of plant growth, thus reducing on-site heat accumulation. Besides, laser beams can be tailored to match the absorption profiles of different plants. We have developed a prototype laser growth chamber and demonstrate that laser-grown plants can complete a full growth cycle from seed to seed with phenotypes resembling those of plants grown under LEDs. Importantly, the plants have lower expression of proteins diagnostic for light and radiation stress. The phenotypical, biochemical and proteomic data show that the singlewavelength laser light is suitable for plant growth and therefore, potentially able to unlock the advantages of this next generation lighting technology for highly energy-efficient horticulture. Furthermore, stomatal movement partly determines the plant productivity and stress management. Abscisic acid (ABA) induces stomatal closure by promoting net K+-efflux from guard cells through outwardrectifying K+ (K+ out) channels to regulate plant water homeostasis. Here, we show that the Arabidopsis thaliana guard cell outward-rectifying K+ (ATGORK) channel is a direct target for ABA in the regulation of stomatal aperture and hence gas exchange and transpiration. Addition of (±)-ABA, but not the biologically inactive (−)-isomer, increases K+ out channel activity in Vicia faba guard cell protoplast. A similar ABA-modulated K+ channel conductance was observed when ATGORK was heterologously expressed in human embryonic kidney 293 (HEK-293) cells. Alignment of ATGORK with known PYR/PYL/RCARs ABA receptors revealed that ATGORK harbors amino acid residues that are similar to those at the latchlike region of the ABA-binding sites. In ATGORK, the double mutations K559A and Y562A at the predicted ABA-interacting site impaired ABA-dependent channel activation and reduced the affinity for ABA in vitro.

Abiotic stresses significantly reduce crop yield, causing serious problems in agriculture. Understanding the mechanisms of plant stress responses could contribute to the improvement of their stress tolerance. Phytohormones play an important role in plant stress defence as well as in regulation of growth and development. This thesis summarizes the results published in four articles focused on the evaluation of the effects of phytohormones during abiotic stresses, namely salinity, drought, ZnO nanoparticle treatment and cold stress. The main emphasis is put on abscisic acid as the key regulator of water status and stress defence, and on cytokinins, which regulate plant growth and stabilize photosynthetic machinery. Cytokinins act antagonistically to abscisic acid. Our results showed that abscisic acid is a general abiotic stress response regulator. Stress- tolerant plants (halophyte Thellungiella salsuginea or winter line of einkorn wheat Triticum monococcum) had a higher basal level of this hormone, especially in shoot meristematic tissues (apices, crowns), than stress-sensitive plants. Stress-tolerant plants reacted faster and in a more flexible way to stress. Active cytokinins were negatively affected by stress, which was associated with growth suppression. The drought stress study showed that...

The effects of temperature, defoliation, light, and certain fall cultural practices on rest and hardiness of Gleason Elberta peach buds were studied. The influence of these factors on abscisic acid concentration in peach buds and a possible role of ABA in rest and hardiness of peach buds were also investigated. Peach leaf buds enter rest in early fall, reach a rest intensity peak in early winter, then break rest as a chilling requirement is met. Complete defoliation before rest has begun will cause some leaf and flower buds to grow, while defoliation after rest has begun will not cause visible bud growth. Effective chill-unit accumulation in the fall apparently did not begin until after a certain amount of leaf abscission had occurred. A GA3 application on August 15, 1974, delayed leaf abscission, and also caused an extension of the rest period. Early fall defoliation was correlated with a reduction in rest intensity and a reduction in the rest period of leaf buds. A result of this study indicates the possibility of the existence of a rest promoting substance that was apparently translocated from a side of a tree exposed to ambient temperatures to a warm greenhouse covered side. Decreasing temperatures caused an increase in flower bud hardiness, however, other factors also had effects on hardiness. An extended day-length treatment caused flower buds to acclimate more slowly than those on untreated trees during the early fall period. The light affect was not evident as colder temperatures prevailed. Buds on trees that were kept warm, acclimated to the same level as buds on trees kept at cooler but non freezing temperatures. However, it took the warm buds approximately four months longer to acclimate. Buds on trees that were exposed to below freezing temperatures acclimated to a much lower level than those on trees not exposed to freezing temperatures. Fall pruning and heavy fertilization with ammonium nitrate did not reduce cold hardiness enough to be measured. However, buds on vigorous, large diameter twigs were more hardy during the early winter period than buds on twigs of smaller diameter. The August 15 and September 19 GA3 treated trees and the early defoliated trees had a delay in acclimation during the fall period, although they did eventually acclimate to the same level as the untreated buds. Abscisic acid concentration in peach leaf and flower buds was low before rest began, increased sharply during the rest inception period, and decreased in concentration before the end of rest. ABA may be a controlling factor in the inception of rest in peach. There was no apparent relationship between ABA concentration and cold hardiness of Gleason Elberta peach flower buds.

La fitohormona ácido abscísico (ABA) juega un papel crucial en el control de la respuesta a estrés y en la regulación del crecimiento y desarrollo de la planta. La unión del ABA a los receptores intracelulares PYR/PYL/RCAR conlleva la inhibición de las PP2Cs del clado A tales como ABI1 o HAB1, causando la activación de la ruta de señalización del ABA. Para obtener más información en la señalización del ABA nos hemos centrado en la caracterización de miembros de estas dos familias proteicas. Hemos generado una versión mutada de HAB1 que contiene una mutación en el Trp-385, residuo clave para la interacción con los receptores y con la molécula de ABA. Como resultado, hab1W385A se mostró refractaria a la inhibición por los receptores PYR/PYL/RCAR. Así, en ensayos de actividad quinasa in vitro encontramos que hab1W385A era capaz de desfosforilar a OST1 incluso en presencia de ABA y de los receptores. hab1W385A y hab1G246D pueden ser clasificadas como mutaciones dominantes hipermórficas. Mientras que hab1G246D posee una actividad fosfatasa reducida, el nuevo alelo dominante muestra una actividad idéntica al genotipo salvaje. Líneas transgénicas de Arabidopsis sobreexpresando hab1W385A mostraron una fuerte insensibilidad al ABA. También hemos analizado el papel de las PP2Cs del clado A pertenecientes a la rama representada por PP2CA. La generación de un mutante doble pp2ca-1hai1-1, que muestra mayor sensibilidad a la hormona en comparación con el genotipo salvaje y con los mutantes sencillos, reveló que HAI1 es un regulador negativo de la ruta de señalización del ABA. El análisis de la localización subcelular mostró que tanto HAI1 como PP2CA se localizan en el núcleo, aunque también están presentes en el citosol y en la fracción microsomal. Tres miembros de la rama de PP2CA i.e.: PP2CA, AHG1 y HAI1, mostraron una inhibición selectiva por los receptores PYR/PYL/RCAR. Estos resultados sugieren que estos receptores pueden discriminar entre miembros del clado A de las PP2Cs. pyl8 es el único mutante sencillo que muestra sensibilidad reducida al ABA en ensayos de crecimiento de raíz. Análisis usando el gen reportero GUS mostraron que PYL8 estaba presente en la estela, en la epidermis de la raíz y en la caliptra, y la cuantificación de la actividad beta-glucuronidasa en raíz mostró que PYL8 es uno de los receptores con mayor nivel de expresión. La caliptra juega un papel crucial en la respuesta hidrotrópica. El estudio de esta respuesta en mutantes múltiples de las PP2Cs y de los PYR/PYL/RCAR reforzó la idea de que el ABA regula este proceso. Así, mientras el mutante séxtuple pyr/pyl112458 presentó una curvatura menor al aplicársele un gradiente de humedad, el mutante cuádruple de las PP2Cs (Qabi2-2) mostró una curvatura más pronunciada en estas condiciones, evitando las zonas con menor potencial hídrico. Finalmente, en la última parte de este trabajo se utilizaron abordajes genético-químicos para aumentar la resistencia a la sequía. Hemos llevado a cabo un rastreo con compuestos químicos para aislar nuevos agonistas del ABA. Basado en datos estructurales de los receptores, se seleccionaron 500 compuestos que fueron ensayados en Arabidopsis. De estos, el compuesto 2C06 inhibió el crecimiento de raíz en plantas salvajes más que en mutantes pyr/pyl/rcar insensibles a ABA y produjo resultados prometedores in vitro al inhibir a las PP2Cs e interaccionar con éstas en ensayos de doble híbrido. / Antoni Alandes, R. (2013). Molecular and genetic analyses of the PP2C-ABA receptor interaction in the abscisic acid signaling pathway [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/29756 / TESIS / Premios Extraordinarios de tesis doctorales