<p>Plant growth and development are strictly regulated by internal hormonal signaling networks, which integrate and coordinate to promote plants’ adaptation and survival in the changing environment. Among the diverse hormones, gibberellins (GAs) are the phytohormones that regulate various processes, from seed germination to fruit development. The conserved plant-specific GRAS family protein DELLAs, key repressors in the GA signaling pathway, serve as the central coordinator of multiple signaling networks through physical interactions with many key transcription factors/regulators in other pathways. </p><p>Diverse DELLA-interacting proteins (DIPs) from different signaling pathways and various protein families have been identified in recent years. All the DIPs interact with the C-terminal GRAS domain of DELLA, however, the mechanism of how the GRAS domain interacts with diverse proteins remains a mystery. To solve this problem, I expressed a number of DELLA proteins in E.coli and obtained high-purity protein for biochemical and structural analysis.</p><p>As the central coordinator of plant growth and development, DELLA’s activity and stability are regulated by post-translational modifications. Our lab recently showed that SECRET AGENT (SEC) modulates the activity of DELLA through O-linked N-acetylglucosamine (O-GlcNAc) modification in Arabidopsis. Nevertheless, SEC’s paralog SPINDLY (SPY), a putative O-GlcNAc transferase (OGT) identified 20 years ago, does not have OGT activity, and serves as opposite role to SEC in GA signaling with an unknown mechanism. </p><p>Our lab made the breakthrough in uncovering the SPY function, and showed it promotes the O-fucosylation of DELLA in planta. I further proved that SPY is a novel protein O-fucosyltransferase through biochemical analysis. SPY specifically transfers O-fucose from GDP-fucose to its substrate peptide, and SPY mutant proteins showed reduced or abolished transferase activity. This is the first work to identify O-fucosylation of nuclear proteins in any organism. O-fucosylation of DELLA activates DELLA by promoting its interaction with DIPs, opposite to repression of interaction with O-GlcNAcylation. Previous studies showed that SPY is involved in multiple cellular pathways such as GA signaling, cytokinin signaling and the circadian clock. Thus, SPY plays an important role in regulating plant growth and development through O-fucosylation of key components in diverse intracellular pathways. </p><p>SPY orthologs are conserved in bacteria, protists, algae and plants, while SEC orthologs are also present in fungi and animals. SPY-like and SEC-like proteins share high sequence similarity, except that two key residues important for the OGT activity of SEC is missing in SPY. Structure analysis of SPY (or its orthologs) would greatly facilitate our understanding of its unique substrate specificity. Toward this goal, I expressed Arabidopsis SPY proteins (as well as bacterial SPY orthologs) in E.coli and obtained high-purity protein for structural analysis. I further identified lead conditions that produce needle-cluster crystals. While optimization would be required, these studies will ultimately reveal the structure of SPY and the architecture of the active site, to show how SPY interacts with GDP-fucose for the transferase activity.</p> / Dissertation

La uva de mesa es la fruta más exportada en Chile. Sobre ella recaen una serie de condiciones que debe cumplir para poder ser exportada, como por ejemplo, que posea un alto calibre de bayas (gran tamaño) y apirenia (ausencia de semillas) en sus frutos. Sin embargo, el desarrollo simultáneo de ambas características es incompatible de forma natural, principalmente por motivos hormonales. Esta memoria se desarrolló en el marco del proyecto Genoma II en uva de mesa, que busca el desarrollo de marcadores genéticos que permitan la identificación temprana de los caracteres antes mencionados. El presente trabajo tuvo como principal objetivo el estudio de la relación entre estos caracteres (apirenia y tamaño de baya) y los perfiles de expresión de los genes de la proteína inhibidora de pectin metilesterasa (PMEI) y de Spindly (SPY). Ambos genes fueron identificados previamente en una búsqueda realizada utilizando el método de mapeo por QTL (Quantitative Trait Locus) y estarían estrechamente ligados con la condición de apirenia estenoespermocárpica y la expresión del carácter tamaño de baya en Vitis vinifera. En este Trabajo de Memoria se diseñaron partidores para aislar por PCR fragmentos de tres secuencias génicas de PMEI (1, 2 y 4) y tres secuencias génicas distintas de SPY (A, B y C) en el genoma de Vitis vinifera, los que se caracterizaron parcialmente en su secuencia nucleotídica y aminoacídica. Se identificó, caracterizó y analizó el patrón de expresión de estos genes, mediante el uso de la técnica de PCR cuantitativo en tiempo real. En este estudio se utilizaron cuatro individuos segregantes con fenotipos contrastantes, en tamaño de baya y presencia/ausencia de semilla, en siete diferentes estadíos de desarrollo. Dichos individuos provienen de la recombinación de dos parentales apirenos estenoespermocárpicos (Ruby Seedless y Thompson Seedless). Adicionalmente, se analizó la posible participación de estos productos protéicos en vías metabólicas, encontrándose que SPYpodría estar participando en la vía de las giberelinas, actuando sobre represores e inhibidores de GA, y PMEI participaría en la vía de la degradación de pectinas inhibiendo el accionar de PME. Al comparar los perfiles de expresión entre individuos con fenotipos contrastantes de baya y semilla, se observó que a medida que las bayas se van desarrollando, ocurren cambios de los niveles de expresión en la mayoría de los genes analizados. Se detectó que nuevos genes identificados en este estudio como PMEI-1 y SPY-C,estarían relacionados con el desarrollo de la baya, mientras que SPY-B estaría asociado a los cambios relacionados con el tamaño de la semilla. En cambio, los dos genes candidatos obtenidos de los estudios por QTL (PMEI-2 y SPY-A), no presentaron cambios en su perfil de expresión que se puedan asociar al fenotipo tamaño de baya o semilla. Este resultado, presenta una inconsistencia con respecto a lo indicado por el método de mapeo por QTL y por ende, se recomienda realizar un nuevo mapeo por QTL con una mayor resolución aumentando el número de marcadores moleculares. Los resultados de esta investigación generaron información genética y biológica de ambos genes, lo que permitirá en el futuro realizar una aproximación hacia el conocimiento de los caracteres en estudio. Sin embargo, se sugiere un nuevo análisis con todas las isoformas génicas encontradas en este estudio, en el que se diferencien tejidos (de baya, de piel y de semilla), y se utilicen a lo menos tres o más segregantes por fenotipo, con el propósito de realizar ensayos con una mayor certeza y precisión a nivel estadístico. Este análisis permitiría obtener datos confiables que relacionen un gen con un fenotipo determinado y no con un individuo en particular.

TOOTH/MIR160A regulates leaf margin outgrowth in Arabidopsis thaliana Unlike animals, a striking aspect of the plant development is that they have evolved a flexible pattern of post embryonic development. This exposes them to the challenges of many biotic and abiotic signals throughout their life. So, plants have to evolve/regulate various mechanisms to modulate their growth and development for accomplishing a successful life cycle in the prevailing environmental conditions. Auxin is involved in the initiation of lateral organs at the meristem and serration development along the leaf margin (Bilsborough et al., 2011, Hay et al., 2006). These two developmental mechanisms share common molecular players. For example, CUC2 is required for the boundary formation at the SAM and also is shown to be essential for serration formation at the leaf margin. Similarly, tth shows increased leaf serration phenotype as well as defects in the positioning of flowers at the meristem. This demonstrates the functional significance of TTH-regulated ARFs in controlling auxin mediated developmental pathways. Leaves originate as small lumps of undifferentiated cells at the flanks of the shoot apical meristem which undergo several rounds division and expansion to generate the mature leaf with characteristic size, shape and leaf margin. Both, endogenous as well as environmental factors modulate the growth and development of a leaf. This is evident from the plasticity in leaf form, observed during the life time of a single plant, as well as from the diversity among closely related species living in different habitats. It is well known that pathways controlling leaf form are subjected to the effects of selection and adaptation. Leaf margin is a key feature of the final leaf shape and it contributes to the abundant diversity in leaf form. Leaf margin architecture varies quite significantly from smooth or entire margin to margins with large outgrowths (lobed margins). The evolution and ecological advantages of this diversity is a subject of intense investigation. It also provides a wonderful system to study the mechanistic details of iterative generation of repeated units, which is a common feature in producing many biological shapes. Recent advances in molecular technologies and the availability of genomic resources ushered the identification of new factors involved in leaf margin development. Our current knowledge of this developmental programme is that CUC2 establishes auxin maxima at the leaf margin by reorienting an auxin efflux carrier PIN1 which ultimately results in serration outgrowth (Bilsborough et al., 2011, Hay et al., 2006). A few missing links in this pathway are the mechanistic details of CUC2 function in reorienting PIN1 and the molecular details of auxin mediated serration outgrowth. Forward genetic screens have been valuable in characterizing a genetic pathway even in the post genomic era. An EMS mutagenesis screen was performed in this context to identify novel factors that can improve our understanding of this intricate mechanism. tooth was identified in the M2 population based on its increased leaf serration phenotype. Genetic analysis showed that tth phenotype is due to a monogenic recessive mutation. Along with increased leaf serration, tth also shows various developmental defects such as aberrant phyllotaxy, narrower cotyledons and narrower leaves. Positional cloning and sequencing analysis showed a G to A transition at the AT2G39175 locus which codes for MIR160A. The mutation is at the 7th base position of the mature miRNA sequence. Functional characterization of miRNAs by isolating mutations is hampered by their small genomic sizes. Till now, only a few miRNAs have been characterized by mutational analysis in plants (Allen et al., 2007, Baker et al., 2005, Cartolano et al., 2007, Chuck et al., 2007, Knauer et al., 2013, Nag et al., 2009, Nikovics et al., 2006). miR160-ARF10 regulatory module is shown to be required for leaf blade out growth and serration, but not leaf complexity in tomato (Hendelman et al., 2012). miR160 is coded by 3 loci in Arabidopsis, MIR160A, B and C. All three loci encode identical mature miRNA that targets 3 Auxin response factors, ARF10, 16 and 17. ARFs are the effector molecules of auxin mediated developmental programmes. Genetic analysis showed that enhanced serration outgrowth in tth is due to the up-regulation of its target genes. Here, we have identified a miRNA that negatively regulates serration outgrowth by repressing ARF10, 16 and 17 whose functional significance in regulating leaf margin development was not known previously. Extensive genetic interaction studies have shown that TTH acts in parallel to SAW-BP and MIR164-CUC pathways in regulating leaf margin development. We have also shown that CUC2 and PIN1 are absolutely essential for serration development in tth. CUC2 establishes a pattern required for the expression of ARF10 at the leaf margin. In the absence of CUC2, downstream effector molecules such as ARFs can not perform their function. arf10-2 arf16-2 could reduce, but not suppress serration outgrowth in various mutants suggesting their functional redundancy with other ARF family members. CUC2 establishes auxin maxima at the leaf margin that triggers the degradation of AUX/IAA repressors thereby relieving ARF proteins which mediate serration outgrowth. Whereas, TTH acts at the post transcriptional level for maintaining normal ARF transcript levels Role of SPYINDLY in Arabidopsis leaf margin development SPYNDLY encodes an O-linked N-acetyl glucosamine transferase that acts as a negative regulator of GA response. Consistent with its role in GA response, spy mutants show several GA dependent phenotypes such as early flowering and hyper branched trichomes. spy mutants also show several GA independent phenotypes such as aberrant phyllotaxy and smooth leaf margin. We have studied its role in regulating Arabidopsis leaf serration development. Reporter analysis of ARF10::GUS and CUC2::GUS in spy-3 revealed that SPY is not involved in establishing serration pattern. The spy-3 leaves did not show any defects during the early stages of serration development, but the mature leaves display smooth leaf margin indicating that SPY function is required for serration outgrowth. As shown in the present study, TTH regulated ARFs are also involved in serration outgrowth. Analysis of leaf margin phenotype in tth spy-3 showed that SPY activity is not required for ARF mediated serration outgrowth. Similar genetic interaction studies with SAW-BP pathway mutants showed that leaf margin out growth mediated by meristematic genes is not dependent on SPY function. Genetic interaction studies with MIR164-CUC pathway genes showed that SPY is required for serration outgrowth in these mutants. Interestingly, the cuc2-3 mutant is defective at both patterning and outgrowth of serration. The spy-3 could suppress serration out growth in cuc2-D suggesting that CUC2 mediated serration out growth is dependent on SPY activity. Protein-protein interaction studies between SPY and CUC2 are in progress to demonstrate whether SPY directly interacts with CUC2 or CUC2 derived signal to regulate serration out outgrowth. It is interesting to examine how mutations at SPY locus can abolish serration out growth mediated by CUC2, but does not affect the serration pattern, even though CUC2 is reported to be essential for both the patterning and outgrowth of serration.