Please read the abstract in the dissertation / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

Iron is an essential micronutrient that plays a role in essential processes in all living organisms. Because iron deficiency anemia is the number one human nutritional deficiency worldwide, research has been focused on studying biofortification, a method of plant breeding focused on increasing the nutrient content of the grain. In order to effectively implement this strategy, research to understand the molecular mechanisms surrounding iron uptake and maintenance within plants is necessary. A major goal of this work lies in exploring iron signaling in Arabidopsis thaliana and further characterization of the yellowstripe1-like1yellowstripe1-like3 (ysl1ysl3) double mutant plant. As shown here, iron signaling in arabidopsis appears to be regulated by a long distance signal derived in the shoots. The ysl1ysl3 double mutant is a plant shown to have severe mis-regulation of iron regulated genes. Here, I show ysl1ysl3 mutant plants are unable to respond to iron deficiency properly and lose signaling ability. It has been proposed that these defects are due to iron accumulation in the shoots but that appears untrue suggesting alternative functions for YSL1 and YSL3 in iron signaling. Another goal of the work included here seeks to elucidate alternative roles for ZmYS1 in maize outside of primary acquisition of iron from the soil. Here, ZmYS1 expression is shown via promoter::GUS analysis expressing throughout the shoot in many cell-types consistent with a role in the movement of iron between leaf tissues. Interestingly, expression in germinating seeds suggests ZmYS1 may also play a role in remobilization of stored iron in developing embryos.

Understanding the causal relationship between genotype and phenotype is a major objective in biology. The main interest is in understanding trait architecture and identifying loci contributing to the respective traits. Genome-wide association mapping (GWAS) is one tool to elucidate these relationships and has been successfully used in many different species. However, most studies concentrate on marginal marker effects and ignore epistatic and gene-environment interactions. These interactions are problematic to account for, but are likely to make major contributions to many phenotypes that are not regulated by independent genetic effects, but by more sophisticated gene-regulatory networks. Further complication arises from the fact that these networks vary in different natural accessions. However, understanding the differences of gene regulatory networks and gene-gene interactions is crucial to conceive trait architecture and predict phenotypes. The basic subject of this study – using data from the Arabidopsis 1001 Genomes Project – is the analysis of pre-mature stop codons. These have been incurred in nearly one-third of the ~ 30k genes. A gene-gene interaction network of the co-occurrence of stop codons has been built and the over and under representation of different pairs has been statistically analyzed. To further classify the significant over and under- represented gene-gene interactions in terms of molecular function of the encoded proteins, gene ontology terms (GO-SLIM) have been applied. Furthermore, co- expression analysis specifies gene clusters that co-occur over different genetic and phenotypic backgrounds. To link these patterns to evolutionary constrains, spatial location of the respective alleles have been analyzed as well. The latter shows clear patterns for certain gene pairs that indicate differential selection. / Das Verständnis des kausalen Zusammenhangs zwischen Genotyp und Phänotyp ist ein wichtiges Ziel in der Biologie. Das Hauptinteresse liegt darin, die Merkmalsarchitektur zu verstehen und Loci zu identifizieren, die zu den jeweiligen Merkmalen beitragen. Genome-wide association mapping (GWAS) ist ein Werkzeug, um diese Zusammenhänge aufzuklären und wurde erfolgreich in vielen verschiedenen Arten eingesetzt. Die meisten Studien konzentrieren sich jedoch auf marginale Markereffekte und ignorieren epistatische und Gen-Umwelt-Interaktionen. Diese Wechselwirkungen sind problematisch zu erklären, werden aber wahrscheinlich einen wichtigen Beitrag zu vielen Phänotypen leisten, die nicht durch unabhängige genetische Effekte, sondern durch ausgefeiltere genregulatorische Netzwerke reguliert werden. Eine weitere Komplikation ergibt sich aus der Tatsache, dass sich diese Netzwerke in verschiedenen natürlichen Akzessionen unterscheiden. Das Verständnis der Unterschiede zwischen genregulatorischen Netzwerken und Gen-Gen- Interaktionen ist jedoch entscheidend, um die Merkmalsarchitektur zu konzipieren und Phänotypen vorherzusagen. Das grundlegende Thema dieser Studie – unter Verwendung von Daten aus dem Arabidopsis 1001 Genomes Project – ist die Analyse von vorzeitigen Stop-Codons. Diese sind in fast einem Drittel der ~ 30k-Gene aufgetreten. Ein Gen-Gen- Interaktionsnetzwerk des gleichzeitigen Auftretens von Stop-Codons wurde aufgebaut und die Über- und Unterrepräsentation verschiedener Paare wurde statistisch analysiert. Um die signifikante über- und unterrepräsentierte Gen-Gen-Interaktion in Bezug auf den biologischen Prozess der kodierten Proteine weiter zu klassifizieren, wurden genonkologische Begriffe (GO-SLIM) verwendet. Darüber hinaus spezifiziert die Koexpressionsanalyse Gencluster, die über verschiedene genetische und phänotypische Hintergründe hinweg gleichzeitig auftreten. Um diese Muster mit evolutionären Einschränkungen in Verbindung zu bringen, wurde auch die räumliche Lage der jeweiligen Allele analysiert. Letzteres zeigt klare Muster für bestimmte Genepaare, die auf eine differentielle Selektion hinweisen.

This study shows that Arabidopsis thaliana WRKY45 gene has an important role in limiting green peach aphid (GPA; Myzus persicae Sülzer) infestation. WRKY45 belongs to the WRKY family of transcription factors, which is one of the largest transcription factor family in plants. In response to GPA infestation, expression of WRKY45 was systemically upregulated in leaves and roots, with highest expression in the vascular tissues, which are the site of aphid feeding. GPA colonization was better on the wrky45 mutant compared to the wild-type (WT) plant. In contrast, GPA poorly colonized plants that were overexpressing (OE) WRKY45, thus confirming an important role for WRKY45 in plant defense to the GPA. A WRKY45-dependent process adversely impacted the reproductive rate of GPA and feeding from the sieve elements. RNA-seq experiments indicated a major impact of WRKY45 overexpression on expression of genes associated with dehydration and abscisic acid biosynthesis and signaling. In agreement with the RNA-seq data, ABA content was also higher in WRKY45-OE plants. However, genetic studies with an ABA-insensitive mutant (abi2-2) indicates that the WRKY45-OE conferred resistance to GPA is mediated through an ABA-independent mechanism. WRKY45-OE plants showed enhanced tolerance to drought and salt stresses. Genetic studies indicate that ABA signaling is critical for WRKY45's involvement in promoting plant tolerance to drought. Taken together, these results demonstrate that WRKY45 acts as a positive regulator of plant responses to GPA infestation, and drought and salt stress responses.

No description available.

No description available.

The Arabidopsis genome encodes 17 putative beta-galactosidases belonging to Glycosyl Hydrolase (GH) family 35, which have been classified into seven subfamilies based on sequence homology. The largest of these, Subfamily III, consists of six genes, Gal-1 (At3g13750), Gal-2 (At3g52840), Gal-3 (At4g36360), Gal-4 (At5g56870), Gal-5 (At1g45130), and Gal-12 (At4g26140) that share 60-81% sequence identity at the amino acid level. All six proteins have a signal peptide that may target them to the cell exterior. We report purification and biochemical characterization of all six members of Subfamily III, each expressed as a recombinant protein in Pichia pastoris and one also in native form, purified from Arabidopsis leaves, with a special emphasis on substrate specificities. Organ specific expression of the six Gal genes was examined by analysis of the microarray databases and by semi-quantitative RT-PCR. The relative abundance and size of the Gal-1, Gal-2, Gal-5, and Gal-12 proteins was studied by immunoblotting using isoform-specific anti-peptide antibodies. The protein expression patterns of the Gal genes were generally consistent with microarray and RT-PCR data, though some discrepancies were observed suggesting distinct mechanisms of regulation for transcription and translation. Localization of total beta-galactosidase activity was visualized using the substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galatopyranoside (X-Gal), to stain whole plants. Subcellular localization of the four isoforms examined by immuno-dotblotting and western blotting showed that Gal-1, Gal-2, Gal-5 and Gal-12 are present in apoplastic and cell wall bound protein extracts. Immuno-EM analysis of Gal-1 and Gal-12 showed that these proteins are localized in the cell walls of vascular and epidermal tissues in mature root. Taken together, the biochemical properties, expression patterns, and subcellular localization of these isozymes indicate that the Subfamily III beta-galactosidases all have potential functions in restructuring the cell wall during plant growth and development. / Ph. D.

Developing oilseeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated at the transcriptional level through the transcription factors belonging to the LAFL regulatory network. The VAL genes encode repressors of the seed maturation program in germinating seeds, but they are also expressed during early stages of seed maturation. VAL1 was identified through a reverse genetics approach as a regulator of seed metabolism, as val1 mutant seeds accumulated elevated levels of storage proteins compared to the wild type. Two VAL1 splice variants were identified, yielding the canonical protein and a truncated protein lacking the plant-homeodomain-like domain important for epigenetic repression. Transcriptomics analysis also revealed that VAL1 is a global epigenetic and transcriptional repressor in developing embryos, though none of the transcripts encoding the LAFL network regulators, including FUSCA3, were affected in val1 embryos. However, VAL1 action is connected specifically to FUSCA3 as 38% of transcripts belonging to the FUSCA3 regulon, but not to other regulons, were largely de-repressed in the absence of VAL1. Based on our model, FUSCA3 activates expression of VAL1 to repress transcription of seed maturation genes without interfering with expression of the core LAFL regulators. / Ph. D.

El desarrollo xilemático es un aspecto fuertemente regulado por hormonas y otros factores de tipo endógeno. La caracterización el mutante acaulis5 (acl5) en Arabidopsis thaliana, deficiente en la síntesis de termoespermina, permitió asignar a esta poliamina una función como regulador de la maduración del xilema, impidiendo la muerte celular antes de que el proceso de diferenciación haya culminado. Aunque se desconoce el mecanismo molecular concreto, se ha identificado un grupo de factores de transcripción de la familia bHLH (SAC51/AJAX1, AJAX2, AJAX3 y AJAX4) cuya traducción es promovida por la termoespermina y es imprescindible para la correcta maduración del xilema, y que actúan como represores de la actividad de LONESOME HIGHWAY (LHW) con el fin de controlar temporalmente los eventos de diferenciación dirigidos por este factor de transcripción. Aunque se sabe que la degradación de las proteínas DELLAs por parte de las giberelinas (GAs) es necesaria para el aumento de crecimiento secundario que acompaña la transición floral, se desconoce el mecanismo molecular concreto por el que esto sucede. En un rastreo de doble híbrido en levadura para la identificación de factores de transcripción que median la actividad de las proteínas DELLA se identificó AJAX3 como interactor de GAI, por lo que el objetivo de esta tesis ha sido el de comprobar si las giberelinas (GAs) regulan la maduración del xilema, y si lo hacen a través de esta interacción. El análisis genético, fisiológico y molecular ha demostrado que: (1) la deficiencia en GAs provoca un fenotipo en el xilema similar al de la falta de función de ACL5; (2) es posible localizar a las DELLAs en la vasculatura, solapando con la expresión de otros elementos necesarios para la diferencación del xilema, como ACL5 y LHW; y (3) la acumulación de DELLAs específicamente en el dominio de expresión de ACL5 en la vasculatura dificulta la maduración del xilema. Además, AJAX3 inhibió la capacidad de LHW de activar a sus dianas en ensayos de expresión transitoria en Nicotiana benthamiana, y la coexpresión de GAI alivió esta represión. En base a estos resultados, proponemos que las GAs promueven la maduración del xilema al menos a través de la modulación de la actividad de AJAX3 para acomodar el proceso de diferenciación al aumento de crecimiento secundario durante la transición flora / Álvarez Mahecha, JC. (2014). CONTROL DE LA DIFERENCIACIÓN DEL XILEMA POR GIBERELINAS A TRAVÉS DE LA INTERACCIÓN DELLA-AJAX3 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48454

Plant development is an iterative process of organ formation from the primary meristems of the plant. Meristem activity is driven by dynamic transcriptional programs that determine cell fate and identity as cells are displaced trough the meristematic tissue to initiate organ primordia. This regulatory network includes members of the TCP and KNOX family of transcription factors, and integrates external and intrinsic cues to efficiently adapt meristem activity to an ever-changing environment. However, how this integration occurs is not clear yet. DELLA proteins have been proposed to modulate transcriptional circuits in plants in response to environmental signals. Although they do not show DNA binding capacity, DELLAs regulate transcription through physical interaction with a large number of DNA-binding transcription factors and other transcriptional regulators. Given the observed interaction between DELLAs and several members of the TCP family of transcription factors, we have explored the relevance of this interaction in the regulation of primary meristems. We have confirmed that DELLAs interact with members of both Class I and Class II TCPs, and prevent their ability to regulate downstream targets. In the embryonic roots, DELLAs maintain a dormant meristem by impairing TCP14/15-dependent activation of cell-cycle genes. On the other hand, DELLAs participate in the establishment of the shoot apical meristem domain that keeps an indeterminate fate, through the control of KNAT1 gene expression by the TCP2/4-AS1 regulatory module. In summary, this Thesis provides a mechanistic framework to eventually explain environmental regulation of meristem activity. / El desarrollo de las plantas es un proceso iterativo de formación de órganos a partir de los meristemos primarios de la planta. La actividad meristemática está dirigida por programas transcripcionales dinámicos que determinan el destino y la identidad celular conforme las células son desplazadas a través del tejido meristemático para iniciar el primordio del órgano. Esta red regulatoria incluye miembros de las familias de factores de transcripción TCP y KNOX, e integra señales externas e intrínsecas para adaptar eficientemente la actividad meristemática al medio ambiente, siempre cambiante. Sin embargo, la manera en que esta integración ocurre no se ha desvelado todavía. Se ha propuesto que en plantas, las proteínas DELLA modulan los circuitos transcripcionales en respuesta a señales medioambientales. Aunque no muestran capacidad de unión al ADN, las DELLAs regulan la transcripción a través de su interacción física con un gran número de factores de transcripción capaces de unirse al ADN y otros reguladores transcripcionales. Dada la interacción observada entre las DELLA y varios miembros de la familia de factores de transcripción TCP, hemos explorado la relevancia de esta interacción en la regulación de los meristemos primarios. Hemos confirmado que las DELLA interaccionan con miembros de las dos clases de TCPs (Clase I y Clase II) e impiden su capacidad de regular dianas aguas abajo. En la raíz del embrión, las DELLAs mantienen el meristemo durmiente al impedir la activación de los genes de ciclo celular dependiente del módulo TCP14/15. Por otro lado, las DELLAs participan en el establecimiento del meristemo apical del tallo, que mantiene un estado indiferenciado, a través del control el módulo TCP2/4-AS1, el cual regula la expresión del gen KNAT1. En resumen, esta Tesis aporta un marco mecanístico para explicar, con el tiempo, la regulación medioambiental de la actividad meristemática. / El desenvolupament de les plantes consiteix en un procés iteratiu de formació d'órgans a partir dels meristems primaris. L'activitat meristemàtica està diridida per programes transcripcionals dinàmics que determinen el destí i la identitat cel.lular a mesura que les cèl.lules es van allunyant del meristem per formar els primordis d`órgans. Esta xarxa de regulació inclou membres de les famílies de factors de transcripció TCP i KNOX, i integra senyals externes i intrínseques per adaptar d'una manera eficient l'activitat del meristem als canvis del medi ambient. No obstant, no es coneix de quina manera la planta fa esta integració. S'ha proposat que les proteïnes DELLA modulen estes xarxes transcripcionals en resposta a senyals del medi. Estes proteïnes no tenen capacitat d'unir-se a l'ADN, però regulen la transcripció mitjançant la interacció amb factors de transcripció i altres reguladors transcripcionals. Donada la interacció entre les proteïnes DELLA i alguns membres de la família de factors de transcripció TCP, hem explorat la rellevància d'esta interacció a la regulació dels meristems primaris. Hem confirmat que les DELLA interaccionen amb membres de les dos classes de TCPs (Classe I i Classe II) i els impedeixen regular les seues dianes. A l'arrel de l'embrió, les DELLA mantenen el meristem dorment al impedir l'activació de gens del cicle cel.lular depenent del mòdul TCP14/15. Per una altra banda, les DELLA particípen a l'establiment del meristem apical de la tija, al que mantenen en un estat indiferenciat, mitjançant el control del mòdul TCP2/4-AS1, que regula l'expressió de KNAT1. En resum, esta Tesi aporta un marc mecanístic per poder explicar, més endavant, la regulació mediambiental de l'activitat meristemàtica. / Felipo Benavent, A. (2017). Modulation of primary meristem activity by gibberellins through DELLA-TCP interaction in Arabidopsis [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/82237