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Greater projected demand for food, fuel, and fiber will require substantial increases in global agricultural production over the next three decades. Climate change is also forecasted to make weather events more extreme and variable. Efficiency will become more important as demand for food products increases and the availability of fertilizer and land decreases. Technology may be of paramount importance for pushing the boundaries of production while remaining sustainable for generations to come. The first chapter of this dissertation investigated the importance of rate and timing of the plant growth regulator trinexapac-ethyl to malting barley in Virginia. Plant growth regulators can help plants remain upright during strong winds, thereby preserving grain quality and yield. However, this study demonstrated that risks of plant injury also exist. Application should be restricted to fields with greater risk of lodging and made only after the barley crop has broken dormancy and a substantial increase in air temperature is not forecasted in the week following application. Chapter two compared the efficacy of eight vegetation indices calculated from three satellites (Landsat 8, Sentinel 2, and Planet) for estimating cover crop biomass. Cover crops can have beneficial effects on agricultural land as well as groundwater and surface water, but only when adequate biomass is established to reduce erosion and nutrient leaching. Satellite imagery was able to estimate multi-species cover crop biomass more accurately than field-based sensors, although the most accurate vegetation index was dependent upon which satellite was being tested. Chapter three investigated the potential of Arabidopsis thaliana ipk1-, a loss-of-function mutant which exhibits decreased growth at elevated phosphorus concentration, for serving as in indicator of plant available phosphorus. An indicator crop could provide greater spatial resolution compared to soil testing, as well as represent plant available nutrients as opposed to chemically extracted nutrient estimations. Plant response exhibited a quadratic relationship with media P concentration in the range of fertilizer decision making for maize, providing valuable insight for potential yield response in agricultural fields below 'very high' phosphorus concentration. / Doctor of Philosophy / Climate change, increased demand for locally sourced ingredients, and elevated pressure for environmentally responsible practices will make meeting the growing demand for food difficult for farmers to achieve over the next few decades. Similar to many other industries, implementation of advanced technology may be necessary to keep up with agricultural demand. Plant growth regulators are one such technology which when applied to plants can cause them to remain short, decreasing the chance of blowing over during windstorms. However, chapter one of this dissertation concluded that risks of plant injury also exist when applying plant growth regulator on malting barley (for brewing or distilling). Application should be restricted to fields with greater risk of wind damage (e.g. taller barley) and made only after the barley crop begins spring growth and a decrease in air temperature is not forecasted in the week following application. Chapter two compared eight spectral vegetation indices across three satellites with different image resolution for their ability to estimate cover crop biomass. Cover crops protect groundwater and surface water quality, but only when adequate growth is achieved. Satellite imagery was able to estimate multi-species cover crop biomass more accurately than field-based sensors, although the most accurate vegetation index was dependent upon which satellite was being tested. Chapter three investigated the potential of Arabidopsis thaliana ipk1-, a loss-of-function mutant which exhibits decreased growth at elevated phosphorus concentration, as in indicator of plant available phosphorus in soil. An indicator crop could help determine which areas of a field are likely to have increased crop yield if fertilized and which are not. The mutant tested could be useful as an indicator crop given its response to phosphorus concentration, warranting further research with other plant species more appropriate for field use.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

Die Biosynthese von Chlorophyll ist komplex und umfasst mehr als ein Dutzend enzymatische Schritte. Es ist nur allzu selbstverständlich, dass eine Deregulation der Chlorophyllsynthese globale Effekte auf die Zelle hat. Um diese Konsequenzen näher zu beleuchten, wurden Arabidopsis thaliana Pflanzen mit chemisch induzierter Deaktivierung von zwei Chlorophyllbiosynthesegenen (CHLH bzw. CHL27) erzeugt sowie photoautotophe Zellsuspensionskulturen von Arabidopsis thaliana hinsichtlich kurzzeitig induzierter Signalprozesse untersucht. Die Resultate verdeutlichen, dass durch Fehlregulationen innerhalb der Chlorophyllbiosynthese erzeugte reaktive Sauerstoffspezies die Transkriptionskontrolle kernkodierter Gene beeinflussen. Die Untersuchung eines enzymatischen Schrittes der Chlorophyllbiosynthese trat in dieser Arbeit in den Hauptfokus: Die Bildung des fünften, isozyklischen Ringes im Chlorophyllmolekül, katalysiert durch das bisher unzureichend erforschte Enzym Mg-Protoporphyrin-IX-monomethylester-Cyclase (Cyclase). Anhand von transgenen Cyanobakterien und Pflanzen sollte das noch unbekannte Gen ycf54 hinsichtlich seiner physiologischen Funktion in dem Cyclase-Enzymschritt analysiert werden. Das Fehlen von Ycf54 in Synechocystis sp. PCC6803 bzw. des homologen LCAA-Proteins in Nicotiana tabacum und Arabidopsis thaliana führt zu starken Cyclase-Substrat-Akkumulationen, verringerten Chlorophyllgehalten und reduzierten Ycf59- bzw. CHL27-Proteingehalten. Ein Mangel von Ycf54/LCAA beeinträchtigt daher die Funktionalität des Cyclase-Komplexes und scheint sich zudem interessanterweise auch auf die Stabilität photosynthetischer Antennenkomplexe auszuwirken. Mittels Pulldown-Assays konnte für Arabidopsis thaliana die direkte physikalische Interaktion zwischen LCAA und CHL27 bestätigt werden. Darüber hinaus sind erste Hinweise für die Ferredoxin-NADP-Reduktase als potenziellen Interaktionspartner gezeigt. / Synthesis of chlorophyll is a complex metabolic process and encompasses more than a dozen enzymatic reactions. It is self-evident that a deregulation of chlorophyll biosynthesis evokes global cellular impacts. To elucidate these consequences Arabidopsis thaliana plants with chemically inducible deactivation of two chlorophyll biosynthesis genes (CHLH and CHL27, respectively) were generated and photoautotrophic cell suspension cultures of Arabidopsis thaliana were used for short induced signal processes. The results illustrate that reactive oxygen species provoked by a deregulated chlorophyll synthesis affect the control of transcription of nuclear genes. The investigation of one enzymatic step of chlorophyll biosynthesis was placed as main focus: The formation of the isocyclic ring of the chlorophyll molecule catalyzed by the Mg protoporphyrin IX monomethyl ester cyclase (short: cyclase), an enzyme which is not fully investigated so far. The still unknown hypothetical chloroplast open reading frame (ycf) ycf54 should be analyzed concerning it’s physiological function in the enzymatic step of the cyclase using transgenic cyanobacteria and plants. Lack of Ycf54 in Synechocystis sp. PCC6803 and the homologous LCAA protein in Nicotiana tabacum and Arabidopsis thaliana, respectively, leads to chlorophyll deficiency, a strong accumulation of the cyclase substrate and reduced protein contents of Ycf59 and CHL27, respectively. A deficit of Ycf54/LCAA impairs the functionality of the cyclase complex and also might compromise the stability of photosynthetic antenna complexes. Using pull-down assays a direct physical interaction between LCAA and CHL27 could be confirmed. Additionally, first evidences for ferredoxin NADP reductase as a potential interaction partner was given.

La défense pré-invasive ou stomatique est un mécanisme qui consiste en la fermeture des pores stomatiques présents sur les organes aériens des plantes lorsque celles-ci sont en contact avec certains agents pathogènes. Cette fermeture empêche ces derniers de pénétrer dans l’hôte et de le coloniser. Ce mécanisme s’active chez Arabidopsis inoculée par la bactérie Pseudomonas syringae pv tomato (Pst) DC3000. Des travaux préliminaires de notre groupe avaient montré que la carbonylation de protéines cibles par des espèces réactives électrophiles (EREs) représentait une étape cruciale de la signalisation cellulaire nécessaire à la mise en place de cette défense. Par des approches de marquage ciblé et de purifications couplées à des identifications par spectrométrie de masse en tandem (nanoLC-MS/MS), nous avons pu caractériser une sérine-thréonine protéine kinase qui joue un rôle déterminant dans ce mécanisme de défense. En effet, des plantes mutées sur le gène codant cette protéine ont perdu la capacité à induire la fermeture de leurs stomates et à déployer la défense stomatique vis-à-vis de la bactérie. De plus, l’introduction de la chimie click (cycloaddition alcyne-azide catalysée par le cuivre), dans nos approches de marquage, nous a permis d’identifier un ensemble de protéines très probablement carbonylées et susceptibles de jouer un rôle crucial dans ces évènements cellulaires qui contribuent à une part de l’immunité végétale. Enfin, les EREs étant capables d’induire la fermeture des stomates, nous avons cherché à savoir, dans le cadre de l’établissement d’une preuve de concept, si leur application sur des plantes permettrait la protection de ces dernières contre Pst. / Pre-invasive or stomatal defense is a mechanism which consists of closing the stomata present at surface of aerial organs of plants when they are in contact with certain pathogens. This closure prevents them from entering and colonizing the host. This mechanism is activated in Arabidopsis inoculated by the bacterium Pseudomonas syringae pv tomato (Pst) DC3000. Preliminary work by our group had shown that carbonylation of target proteins by reactive electrophile species (RES) was a crucial step of the cell signaling required to set up this defense. Through targeted tagging and purifications approaches coupled with tandem mass spectrometry identifications (nanoLC-MS/MS), we have been able to characterize a serine-threonine protein kinase that plays a crucial role in this defense mechanism. Indeed, plants mutated on the gene encoding this protein have lost their ability to trigger stomatal closure and to deploy the stomatal defense against the bacteria. In addition, the use of the click chemistry and notably, the copper-catalyzed alkyne-azide cycloaddition, in our tagging approaches has enabled us to identify a set of proteins that are most likely carbonylated and likely to play a significant role in these cell events that contribute to part of plant immunity. Finally, since RES are able to induce stomatal closure we sought to find out, in the context of establishing a proof-of-concept, whether their application to plants would enable them to be protected against the Pst.

El trabajo realizado en la Tesis Doctoral llega a las siguientes conclusiones:1.- T. halophila muestra una relación en peso raíz/parte aérea mayor que A. thaliana, y esta diferencia se ve incrementada en condiciones de estrés salino, lo que podría suponer una ventaja para afrontarlo.2.- El estrés salino produce en A. thaliana mayores reducciones en la absorción y en las concentraciones internas de K+ que en T. halophila, a la vez que T. halophila presenta menor absorción de Na+ y transporte a la parte aérea que A. thaliana. Ambas circunstancias resultan en una mayor relación K+/Na+ en T. halophila, lo que puede suponer una mayor tolerancia a la salinidad.3.- El gen ThHAK5 codifica para un transportador que media un transporte de K+ de alta afinidad en levaduras similar al observado en las plantas de T. halophila lo que sugiere que este transportador juega un papel fundamental en la absorción de K+ en el rango de la alta afinidad en esta especie vegetal.4.- Aunque AtHAK5 y ThHAK5 presentan una gran homología de secuencia y unas características funcionales similares, la regulación de los genes que los codifican difieren en condiciones salinas. Así, la salinidad reduce en menor medida la inducción de ThHAK5 por ayuno de K+. En consecuencia, la absorción de K+ de alta afinidad está menos afectada por la presencia de NaCl en el medio externo en T. halophila.5.- La mutagénesis al azar permite encontrar aminoácidos importantes para la función de las proteínas y ésta ha permitido identificar dos versiones mutantes del transportador de K+ de alta afinidad AtHAK5 más eficientes, capaces de transportar K+ a concentraciones externas de Na+ muy elevadas (0.1 mM K+ y 800 mM Na+). / The work done in this Thesis provides some interesting conclusions:1.- Thellungiella halophila show a weight ratio root/shoot bigger than Arabidopsis thaliana, and this difference arise under salt stress, what might provide an effective mechanism of salt tolerance to T. halophila.2.- In A. thaliana, salt stress induces a bigger reduction of K+ uptake and tissue concentrations than in T. halophila, and at the same time T. halophila shows a reduced Na+ uptake and Na+ transport to the shoot. Both properties enable a higher ratio K+/Na+ in T. halophila which might be another mechanism of salt tolerance. 3.- The ThHAK5 gene isolated in this Thesis, encode a K+ transporter that mediates high affinity K+ transport in Saccharomyces cerevisiae similar to the observed in intact plants of T. halophila, which suggest a key role of this transporter in the high affinity range of concentrations.4.- Although AtHAK5 and ThHA5 shows high sequence homology and similar functional properties, gene regulation is different under salt stress. Thus, salinity reduces to a lesser extent the K+-starvation ThHAK5 induction. As a consequence, high affinity K+ uptake is less affected by NaCl in T. halophila. 5.- Random mutagenesis allows the identification of important aminoacids for protein function, and with this technique two more efficient mutant versions of AtHAK5 have been isolated. The evolved AtHAK5 mutant versions are able to transport K+ at high Na+ external concentrations (0.1 mM K+ and 800 mM Na+) in yeast.

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