This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p> Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA. This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA (phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has suggested the selectivity of binding. Further, Rubisco enzymatic activity in the absence of ABA was compared to that in the presence of ABA at various concentrations. The results have clearly indicated the effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate (RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most resembles allosteric inhibition. The biological function of this newly discovered interaction is interpreted as ABAs ability to regulate plant growth during abiotic stress by its direct action on the photosynthetic machinery - hypothesis often suggested in the literature.

This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p> Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA. This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA (phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has suggested the selectivity of binding. Further, Rubisco enzymatic activity in the absence of ABA was compared to that in the presence of ABA at various concentrations. The results have clearly indicated the effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate (RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most resembles allosteric inhibition. The biological function of this newly discovered interaction is interpreted as ABAs ability to regulate plant growth during abiotic stress by its direct action on the photosynthetic machinery - hypothesis often suggested in the literature.

The release of genetically modified, herbicide tolerant canola (<I>Brassica napus</i> L.) genotypes in western Canada has increased interest in the persistence of volunteer canola. <i>B. napus</i> seed may be induced into secondary dormancy in the laboratory, however, little is known of the seedbank ecology and the role of secondary dormancy as a persistence mechanism in this species in the region. The objectives of this research were i) to determine seedbank additions at the time of harvest, ii) determine the role of secondary seed dormancy in seedbank persistence under different management systems, iii) determine the relative importance of factors contributing to secondary dormancy potential and iv) evaluate the role of abscisic acid (ABA) during secondary dormancy induction in <I>B. napus</i>. On farms, average seedbank additions during harvest were approximately 20 times the normal seeding rate of canola. High secondary seed dormancy potential prolonged seedbank persistence in fields, irrespective of tillage system. <I>B. napus</i> exhibited seedling recruitment of a typical summer annual weed where seedling recruitment was only observed in the spring. Seasonal seedling recruitment was the result of two fates: seed death in the shallow seedbank, irrespective of dormancy potential, and increased ungerminability in buried seeds which was related to secondary seed dormancy potential. Among the factors that contribute to secondary seed dormancy potential, genotype was of greatest significance. Seed size was of lesser importance, while the contributions of pre-harvest factors including seed maturity, year, and location were negligible in comparison. Differences in ABA synthesis and the response to ABA application were related to secondary seed dormancy potential and correlated well previous reports linking ABA to seed dormancy. Conclusions that emerge from this research are i) that on some farms, seedbank additions may be lowered by more diligent harvest practices, ii) seedbank persistence of <I>B. napus</i> may be reduced by growing low dormancy genotypes and avoiding seed burial for one year after seedbank establishment and iii) ABA + ABA-glucose ester (ABA-GE) and the ability of seeds to respond to ABA application after seed dormancy induction may potentially be used to identify seed dormancy potential in this species.

The release of genetically modified, herbicide tolerant canola (<I>Brassica napus</i> L.) genotypes in western Canada has increased interest in the persistence of volunteer canola. <i>B. napus</i> seed may be induced into secondary dormancy in the laboratory, however, little is known of the seedbank ecology and the role of secondary dormancy as a persistence mechanism in this species in the region. The objectives of this research were i) to determine seedbank additions at the time of harvest, ii) determine the role of secondary seed dormancy in seedbank persistence under different management systems, iii) determine the relative importance of factors contributing to secondary dormancy potential and iv) evaluate the role of abscisic acid (ABA) during secondary dormancy induction in <I>B. napus</i>. On farms, average seedbank additions during harvest were approximately 20 times the normal seeding rate of canola. High secondary seed dormancy potential prolonged seedbank persistence in fields, irrespective of tillage system. <I>B. napus</i> exhibited seedling recruitment of a typical summer annual weed where seedling recruitment was only observed in the spring. Seasonal seedling recruitment was the result of two fates: seed death in the shallow seedbank, irrespective of dormancy potential, and increased ungerminability in buried seeds which was related to secondary seed dormancy potential. Among the factors that contribute to secondary seed dormancy potential, genotype was of greatest significance. Seed size was of lesser importance, while the contributions of pre-harvest factors including seed maturity, year, and location were negligible in comparison. Differences in ABA synthesis and the response to ABA application were related to secondary seed dormancy potential and correlated well previous reports linking ABA to seed dormancy. Conclusions that emerge from this research are i) that on some farms, seedbank additions may be lowered by more diligent harvest practices, ii) seedbank persistence of <I>B. napus</i> may be reduced by growing low dormancy genotypes and avoiding seed burial for one year after seedbank establishment and iii) ABA + ABA-glucose ester (ABA-GE) and the ability of seeds to respond to ABA application after seed dormancy induction may potentially be used to identify seed dormancy potential in this species.

Plants rely on diverse strategies to regulate water loss during drought. The phytohormone abscisic acid (ABA) is a critical mediator of stomatal closure during water stress in seed plants. Studies in conifers identified diverging strategies in long-term drought of ABA-mediated dynamics, particularly a peaking type dynamic during long term drought in some conifers. Few studies have reported this dynamic in angiosperms, and no study has revealed the mechanism driving declines in ABA levels as drought progresses in peaking type species. To understand peaking type dynamics, we exposed the model peaking type gymnosperm species <i>Callitris rhomboidea</i> and the highly drought resistant evergreen angiosperm <i>Umbellularia californica</i> to controlled long-term drought. We measured leaf water potentials (Ψ_l), stomatal conductance, ABA and the ABA catabolite phaseic acid (PA) levels in potted plants during a prolonged but non-fatal drought. We aimed to determine which of three potential drivers of peaking type dynamic were responsible for this response: (1) increased catabolism of ABA into PA at a threshold Ψ_l , (2) ABA export from the leaf is enhanced under drought, and (3) ABA biosynthesis ceases at a threshold Ψ_l. During long term drought, the evergreen angiosperm species <i>U. californica</i> demonstrated peaking type ABA dynamics like gymnosperms. In both species, PA levels did not increase significantly, in fact, PA levels tracked ABA levels, suggesting that ABA catabolism to PA may be a function of ABA levels. Girdling experiments to determine whether export from the leaf drove declines in ABA levels demonstrated that of the majority of ABA was likely converted to ABA glucose ester (ABA-GE), an inactive storage form of ABA, and exported from shoots during drought. Finally, by rapidly dehydrating branched collected at different timepoints during long-term drought we were able to determine that ABA biosynthesis is completely down regulated in leaves that have been dehydrated beyond leaf turgor loss point. The decline in ABA levels in peaking type species appears conserved across seed plants and is mediated by high export rates in the form of ABA-GE. Future work should assess a more diverse selection of species as well as study long-term drought in less tolerant species to test whether ABA biosynthesis is deactivated in all species once Ψ_ldeclines below turgor loss point.

Master of Science / Department of Agronomy / P. V. Vara Prasad / In many countries producers have been growing varieties of hard white winter (HWW) wheat since decades. The cause of concern is most varieties of HWW wheat are susceptible to pre-harvest sprouting (PHS) which affects grain quality. Environmental conditions like high humidity, precipitation, heavy dew and hormonal activity at physiological maturity stimulate PHS in HWW. To alleviate these conditions research was carried out at KSU. KS01HW163-4, a sprouting tolerance line was crossed with Heyne, a sprout susceptible cultivar. A total of 224 doubled haploid (DH) lines thus produced were phenotyped in the present study through experiments conducted in controlled environments. The objectives of this research were to (i) characterize and phenotype the doubled haploid lines for PHS in controlled environments; (ii) understand the impact of growth environment (high temperature and/or drought) and; (iii) impact of exogenous application of growth hormones on tolerance to pre-harvest sprouting in the parental lines of the doubled haploid population. The phenotypic data collected from this research will be ultimately combined with the genotypic data to identify DNA markers related to PHS tolerance and provide DNA markers for marker assisted selection. Based on my results of the germination percentages from the 224 DH lines, the population was distributed as susceptible, and tolerant to PHS showing a bimodal distribution and X[superscript]2 analysis indicating a complimentary gene action. From the study of the influence of environmental factors on PHS, my results confirmed a definite influence of stress on sprouting. Under optimum temperature (OT), KS01HS163-4 was tolerant to PHS, but at HT and/or drought it became susceptible to PHS. Growth under stressed conditions changed the tolerance levels to PHS. Seed dry-weight, and harvest index were also influenced negatively due to stress. Therefore multi-location tests must be conducted with variable environments to test the stability of a variety to PHS. From the study of the influence of phytohormones on PHS, the results suggest that tolerance to sprouting was seen in seeds from plants sprayed with abscisic acid (ABA) and paclobutrazol (GA-inhibitor) treatments where as those from gibberellic acid (GA) treatment showed susceptibility to sprouting.

La performance d'une plante repose en partie sur sa capacité à capturer l'énergie lumineuse via la croissance foliaire. La littérature souligne deux limitations majeures de la croissance, de nature métabolique ou hydraulique. Nous testons ici l'hypothèse originale que l'importance relative de ces deux limitations est structurée par l'ontogenèse de la feuille chez Arabidopsis thaliana. Nous montrons que la disponibilité en carbone restreint la croissance des jeunes feuilles, tandis qu'une compétition hydraulique entre croissance et transpiration s'accroît au cours de l'ontogenèse. La mise en place de cette limitation hydraulique s'explique par une dégradation de la capacité du xylème et probablement des aquaporines à approvisionner la feuille en eau, malgré une diminution ontogénétique de la transpiration. Cette dernière est la conséquence de l'acquisition progressive de la sensibilité des stomates aux signaux de fermeture, notamment l'obscurité et l'acide abscissique (ABA), hormone induite par la sécheresse. Enfin, nous mettons en évidence une nouvelle composante de la sensibilité stomatique à l'ABA, conservée chez des mutants décrits comme insensibles à cette hormone : l'ABA induit une diminution de la conductance hydraulique foliaire qui abaisse le potentiel hydrique foliaire et in fine la conductance stomatique. Ce mécanisme chez les feuilles développées contribuerait sous stress hydrique à rediriger le flux d'eau vers les feuilles en croissance. Plus généralement, le contrôle des stomates par des mécanismes hydrauliques induits par l'ABA pourrait être une composante majeure de l'ajustement entre offre et demande en eau chez les plantes soumises à un stress hydrique. / In plants, leaf growth is the central process allowing energy capture and space colonization. The literature suggests that leaf growth is predominantly determined by metabolic and hydraulic limitations. Here, we test the original hypothesis that the relative importance of metabolics and hydraulics on the control of leaf growth is organized according to leaf ontogeny in Arabidopsis thaliana. We show that leaf carbon balance limits growth of the young leaves which therefore grow at a slower rate in the nighttime, while a hydraulic limitation gradually establishes in the daytime, when growth and transpiration competes for water. This gradual hydraulic limitation is underlain by a deterioration of leaf venation and probably aquaporins capacity to supply water to the leaf, despite an ontogenetic decrease in transpiration. This decline in transpiration occurs because stomata acquire throughout leaf ontogeny their sensitivity to the major closure signals, including darkness and abscisic acid (ABA), a hormone induced by drought. Finally, we discover a novel component of stomatal sensitivity to ABA, conserved in mutants described as insensitive to ABA in isolated epidermis: ABA induces a decrease in leaf hydraulic conductance which lowers leaf water potential and stomatal conductance according to a hydraulic cascade. Decreasing leaf hydraulic conductance through ABA action in fully expanded leaves would contribute to redirect water flow to the young leaves under water stress. More generally, controlling stomata by ABA-induced hydraulic mechanisms could be a crucial component of the coordination between water supply and water demand in plants under water challenging conditions.

<p>Predmet ovog istaživanja&nbsp; bio je ispitivanje&nbsp;uticaja jona tri te&scaron;ka metala (Ni²⁺, Cu^2+&nbsp;i Cd²⁺)&nbsp;u dve toksične koncentracije u zemlji&scaron;tu na&nbsp;fiziolo&scaron;ke i biohemijske karakteristike&nbsp;<br />odabranih klonova topola, M1, B229 i Pe 19/66.&nbsp;Ispitan je i potencijal odabranih klonova topola&nbsp;da vr&scaron;e fitoekstrakciju-akumulaciju te&scaron;kih&nbsp;metala iz zemlji&scaron;ta u svoje nadzemne delove &scaron;to&nbsp;dovodi&nbsp; do dugoročnog uklanjanja ovih&nbsp;perzistentnih polutanata iz životne sredine.&nbsp;Takođe, ispitan je uticaj te&scaron;kih metala na&nbsp;antioksidantni potencijal, sposobnost&nbsp;<br />neutalizacije slobodnih radikala, aktivnosti&nbsp;antioksidantih enzima kao i&nbsp; na sadržaj&nbsp;slobodnih i konjugovanih poliamina (Put, Spm,&nbsp;Spd),&nbsp; određenih HPLC analizom,&nbsp; i sadržaj&nbsp;biljnih hormona poput indol-3-sirćetne kiseline&nbsp;i&nbsp; abscisinske &nbsp;kiseline,&nbsp; određenih GC/MS&nbsp;analizom, u listovima i korenovima klonova topola.</p><p>&nbsp;</p> / <p>The aim of this study was to estimate and compare phytoextraction capacities of three&nbsp;poplar clones (M1, B229 and Pe 19/66) in soil. Furthermore, the goal was to assess &nbsp;different biological responses among the poplar clones during exposure to different concentration of three heavy metal ions (Ni²⁺, Cu²⁺ i Cd²⁺). In order to track changes in&nbsp; poplars&rsquo;mineral, physiological, biochemical and antioxidant status during the&nbsp; abiotic stress, quantification of physiological properties, free and conjugated polyamines, total phenolics&nbsp; as well as quantification of phytohormones (indol-3-acetic and abscisic acid) was done. Furthermore, assessment of antioxidant potential by tracking radical scavenger capacities (RSC) against DPPH, ABTS, OH and NO radicals and by measuring enzymes activities (SOD, GSH-Px, GPx, GR) in vitro was performed in root and leaves of poplar clones.</p>

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.