Non-targeted metabolite profiling by liquid chromatography-mass spectrometry (LC-MS) was used to determine the metabolite responses of Arabidopsis roots to auxin or ethylene. Crosstalk between these hormones regulates many important physiological processes in plants, including the initiation of lateral root formation and the response to gravity. These occur in part through alterations in the levels of flavonoids, specialized plant metabolites that have been shown to act as negative regulators of auxin transport. However, much remains to be learned about auxin and ethylene responses at the level of the metabolome. LC-MS analysis showed that a number of ions changed in response to both hormones in seedling roots. Although classes of specialized metabolites such as flavonols and glucosinolates change in abundance in response to both auxin and ethylene, there was little overlap with regard to the specific metabolites affected. These data will be integrated with information from transcriptomic and proteomic experiments to develop framework models that connect phytohormones and specialized metabolism with specific physiological processes. Previous studies by imaging techniques have shown that flavonols increase in response to both auxin and ethylene in the root elongation zone, but LC-MS showed that flavonols decreased in abundance in response to these hormones. Therefore a method was developed for targeted metabolite profiling of flavonols in individual root tips by flow injection electrospray mass spectrometry. This method uncovered spatial differences in metabolic profiles that were masked in analyses of whole roots or seedlings, and verified that flavonols increase in response to these hormones in root tips. / Master of Science

The plant hormones gibberellins (GAs) and auxin display overlapping activities in the regulation of multiple developmental processes, including the differential growth that mediates the response to tropic stimuli and the formation of the apical hook. Several mechanisms have been proposed that explain the interaction between these two hormones, such as the regulation of auxin transport by GAs, and the regulation of GA biosynthesis by auxin. GAs are known to exert their action at the transcriptional level by promoting the degradation of DELLA proteins, which in turn interact with numerous transcription factors and modulate their activity. We have identified INDOLE-3-ACETIC ACID METHYLTRANSFERASE 1 (IAMT1) as one of the earliest target genes upregulated after conditional expression of the DELLA protein GAI in Arabidopsis thaliana. In this Thesis, we have addressed two main issues: (1) the contribution of IAMT1 to auxin homeostasis and its biological relevance; and (2) the molecular mechanism by which DELLAs are able to induce the expression of IAMT1. Using combinations of iamt1 loss-of-function mutants and reporter lines for auxin accumulation and activity, we have found that IAMT1 activity is essential for proper generation and maintenance of the auxin gradients that underlie differential growth. According to our results, the role of IAMT1 would be to restrict polar auxin transport especially during the response to tropic stimuli, preventing excessive auxin accumulation in the responding tissues, and IAMT1 exerts this function, at least in part, by inhibiting the expression of the PIN genes, encoding auxin efflux carriers. Regarding the regulation of IAMT1 expression by DELLAs, dissection of the promoter, in silico analysis of putative DELLA partners, and molecular genetic analysis of reporter lines has allowed us to identify two mechanisms with different relevance depending on the environmental conditions, and through different cis elements. In etiolated seedlings, DELLA proteins are recruited by DORNRÖSCHEN (DRN) to the IAMT1 promoter to induce IAMT1 expression. In the light and in a temperature-dependent manner, DELLA proteins inhibit the DNA-binding activity of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and BRI1 EMS-SUPPRESSOR1(BES1), which act as repressors of IAMT1 expression. The work presented here highlights how GAs may affect local accumulation of auxin, being particularly relevant in processes that involve differential growth. / Abbas, M. (2014). The control of auxin homeostasis through the regulation of IAMT1 by DELLA proteins [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/39348

Plant hormones and their gene regulatory networks orchestrate a diverse array of metabolic and physiological changes crucial for growth, development, and environmental responses. Targeting the engineering of hormone signaling networks holds promise for enhancing plant health, crop productivity, and vigor. However, these networks are intricate, featuring negative feedback loops, extensive interconnections between pathways, pleiotropy, and overlapping gene expression. These complexities pose challenges in identifying candidate genes and parsing apart their isolated functions that could be strategically engineered to achieve desired plant phenotypes. Integration of comparative evolution, synthetic biology, and expression analysis facilitates the deconstruction of these networks. Through systems biology approaches data dimensionality can be reduced, enabling the attribution of specific phenotypes to associated genes. Here, I reviewed how the employment of these above-mentioned approaches can aid in the identification of candidate genes involved the regulation of growth and development within specific tissues, and how through synthetic biology we can explore the sequence-function space of candidate genes and their pathway modules. Candidate genes identified through this process can be evaluated through comparative evolutionary approaches, and efficiently tested in synthetic systems for engineering of their molecular functionalities in a high-throughput manner. Here, as a case study, I employ a systems biology approach to identify tissue-specific candidate genes within the auxin regulatory network in soybean shoot development. This method aims to minimize pleiotropy and off-target effects by utilizing expression analysis tissue-specificity score and principal component analysis. I primarily, focused on three pivotal components of the nuclear auxin signaling pathway: Aux/IAA transcriptional repressors, ARF transcription factors, and TIR1/AFB auxin receptors. These components collectively modulate auxin signaling, influencing various growth and environmental responses. I identified genes within the three pivotal components of auxin signaling involved in early shoot architecture development, which has advantages from weed suppression to yield in soybean cultivation. I used a yeast chassis to investigate the function of pleiotropic auxin receptors, which primarily regulate Aux/IAA levels and orchestrate transcriptional changes in response to auxin. I explored whether these receptors modulate auxin response in a concerted fashion, as they are generally not tissue specific. Here, I reported that auxin receptors interact in an epistatic manner to modulate auxin response. This case of study serves as a foundation in engineering plant genotype-phenotype via auxin signaling. / Doctor of Philosophy / Plant hormones are essential for controlling various processes that drive plant growth, development, and responses to the environment. Scientists are exploring ways to engineer the networks that regulate these hormones to improve plant health, boost crop yields, and enhance plant strength. However, these networks are complex, with many interacting parts, making it difficult to identify which genes to modify to achieve specific outcomes in plants. To tackle this challenge, researchers use a combination of approaches, including studying how these networks have evolved, analyzing large amounts of biological data, and using synthetic biology to test and refine their findings. By breaking down the complexity of these networks, they can link specific genes to particular plant traits. Once these genes–trait links are identified, they can be further tested and engineered to optimize plant characteristics. In this study, I focused on the auxin hormone, which plays a key role in numerous aspects of plant growth including soybean shoot development and Arabidopsis root development. I looked at three main components of the auxin regulatory network: Aux/IAA proteins (which act as repressors), ARF transcription factors (which control gene expression), and TIR1/AFB receptors (which detect auxin levels). These components work together to regulate how plants grow and respond to their environment. I identified key genes within these main auxin components that are important for early development of soybean shoots and minimizes off-target effects. This can help improve soybean farming by enhancing weed control and enhancing crop yields Using a synthetic biology yeast system, I studied the function of TIR1/AFB auxin receptors and how this family of receptors interact to perceive auxin and control the levels of Aux/IAA proteins, consequently controlling the plant's growth in response to auxin. I found that auxin receptors work in concert in a way that reduces their overall effect on the plants response to auxin. This research lays the groundwork for future efforts to engineer plant traits by modifying the auxin signaling pathway, which could lead to improved crop performance and resilience.

No description available.

Enquanto as raízes laterais (RL) se desenvolvem a partir da raiz primária, as raízes adventícias (RA) são geralmente formadas em órgãos da parte aérea da planta. As RA podem ser formadas como uma resposta adaptativa a estresses, como ferimentos ou alagamentos e a sua formação também é importante para a propagação vegetativa de espécies economicamente relevantes, que frequentemente dependem da propagação clonal de genótipos elite. A aplicação de hormônios pode estimular o desenvolvimento das RA (DRA), e as auxinas são consideradas os principais hormônios envolvidos nesse processo. Neste estudo, o sistema de plântulas estioladas foi usado em Arabidopsis thaliana para analisar diversos aspectos do DRA. Diferentes tipos de auxinas, naturais ou sintéticas, foram testadas e verificou-se que AIA causou um aumento no número de raízes sem afetar seu comprimento, ANA foi efetivo para o DRA, mas as raízes ficaram pequenas, e altas concentrações de 2,4-D causaram a formação de calos. Através de imunolocalização, um nível elevado de AIA foi detectado nos tecidos do hipocótilo que deram origem ao primórdio radicular. O padrão de expressão de genes potencialmente envolvidos com o enraizamento adventício foi testado por PCR em Tempo Real. O DRA foi marcado essencialmente por aumento na expressão de PIN1, SUR2, GH3.3, GH3.6, ARF8 e IAA28. A expressão dos genes induzidos foi mais estimulada por ANA, seguida de AIA. A expressão de IAA28 aumentou com o DRA, diferente do que foi observado no desenvolvimento de RL. Os receptores de auxinas TIR1/AFB e ABP1 iniciam a sinalização de auxinas na célula pelo controle da expressão gênica, proteólise seletiva e afrouxamento da parede celular. Verificou-se que TIR1 e as proteínas AFBs são importantes para o DRA, mas que estes receptores devem estar exercendo funções redundantes no processo e que ABP1 pode agir complementando a sua ação. Durante a organogênese das RA, TIR1 e AFB2 parecem exercer uma maior influência. As auxinas são transportadas de maneira polar, célula a célula e geralmente dependem de transportadores. Analisamos o DRA em diferentes mutantes deficientes no transporte de influxo e efluxo de auxinas juntamente com construções com genes repórteres, na presença ou ausência de auxina exógena. Uma função essencial foi estabelecida para AUX1 no enraizamento adventício e, embora LAX3 per se não tenha sido chave no processo, este parece agir em conjunto com AUX1. Também observamos que a formação eficiente de RA depende dos transportadores de efluxo PIN, principalmente PIN1, 3 e 7. A adequada fosforilação dos PINs pelas quinases PID, WAG1 e WAG2 e, consequentemente, a direção do transporte, foi igualmente essencial para o estabelecimento das RA. / Lateral roots (LR) develop from the primary root, whereas adventitious roots (AR) are generally formed from above-ground organs. AR can be formed as an adaptive response to stresses, like wounding or flooding, and their formation is also important for efficient vegetative propagation of economically relevant species, which often depend on clonal propagation of elite genotypes. Hormonal application can stimulate AR development (ARD) and auxins are recognized as major hormones involved in this process. Here, the etiolated seedlings system was used in Arabidopsis thaliana to study several aspects of ARD. Different auxin types, natural or synthetic, were tested and it was found that IAA caused an increase in root number without affecting root length, NAA was effective for ARD, but roots remained short and higher levels of 2,4-D caused callus formation. Through immunolocalization, a higher level of IAA was detected in hypocotyl tissues from which the root primordia differentiated. The expression pattern of genes potentially involved in adventitious rooting was tested by Real-Time PCR. ARD was essentially marked by increased expression of PIN1, SUR2, GH3.3, GH3.6, ARF8 and IAA28. The magnitude of expression of induced genes was much stimulated by NAA, followed by IAA. IAA28 expression increased with ARD, differently from what is known for lateral root development. The auxin receptors TIR1/AFB and ABP1 initiate auxin signaling in the cell through changes in gene expression, selective proteolysis and cell wall loosening. We observed that TIR1/AFB are important in ARD but might be playing redundant roles in the process, whereas ABP1 could be complementing their action. During AR organogenesis, TIR1 and AFB2 seemed to exert greater influence. Auxins are transported in a polar, cell to cell way and depend on several transporters. We analyzed ARD in different mutants affected in auxin influx and efflux transporters, coupled with reporter gene constructs, in presence or absence of exogenous auxin. An essential role was established for AUX1 in AR. Although LAX3 per se was not a key player in the process, it seemed to act in conjunction with AUX1. We also observed that efficient formation of AR depends on the PIN efflux transporters, mainly PIN1, 3 and 7. The proper phosphorylation of PINs by the kinases PID, WAG1 and WAG2, and hence the direction of auxin transport, was equally essential for AR establishment.

Enquanto as raízes laterais (RL) se desenvolvem a partir da raiz primária, as raízes adventícias (RA) são geralmente formadas em órgãos da parte aérea da planta. As RA podem ser formadas como uma resposta adaptativa a estresses, como ferimentos ou alagamentos e a sua formação também é importante para a propagação vegetativa de espécies economicamente relevantes, que frequentemente dependem da propagação clonal de genótipos elite. A aplicação de hormônios pode estimular o desenvolvimento das RA (DRA), e as auxinas são consideradas os principais hormônios envolvidos nesse processo. Neste estudo, o sistema de plântulas estioladas foi usado em Arabidopsis thaliana para analisar diversos aspectos do DRA. Diferentes tipos de auxinas, naturais ou sintéticas, foram testadas e verificou-se que AIA causou um aumento no número de raízes sem afetar seu comprimento, ANA foi efetivo para o DRA, mas as raízes ficaram pequenas, e altas concentrações de 2,4-D causaram a formação de calos. Através de imunolocalização, um nível elevado de AIA foi detectado nos tecidos do hipocótilo que deram origem ao primórdio radicular. O padrão de expressão de genes potencialmente envolvidos com o enraizamento adventício foi testado por PCR em Tempo Real. O DRA foi marcado essencialmente por aumento na expressão de PIN1, SUR2, GH3.3, GH3.6, ARF8 e IAA28. A expressão dos genes induzidos foi mais estimulada por ANA, seguida de AIA. A expressão de IAA28 aumentou com o DRA, diferente do que foi observado no desenvolvimento de RL. Os receptores de auxinas TIR1/AFB e ABP1 iniciam a sinalização de auxinas na célula pelo controle da expressão gênica, proteólise seletiva e afrouxamento da parede celular. Verificou-se que TIR1 e as proteínas AFBs são importantes para o DRA, mas que estes receptores devem estar exercendo funções redundantes no processo e que ABP1 pode agir complementando a sua ação. Durante a organogênese das RA, TIR1 e AFB2 parecem exercer uma maior influência. As auxinas são transportadas de maneira polar, célula a célula e geralmente dependem de transportadores. Analisamos o DRA em diferentes mutantes deficientes no transporte de influxo e efluxo de auxinas juntamente com construções com genes repórteres, na presença ou ausência de auxina exógena. Uma função essencial foi estabelecida para AUX1 no enraizamento adventício e, embora LAX3 per se não tenha sido chave no processo, este parece agir em conjunto com AUX1. Também observamos que a formação eficiente de RA depende dos transportadores de efluxo PIN, principalmente PIN1, 3 e 7. A adequada fosforilação dos PINs pelas quinases PID, WAG1 e WAG2 e, consequentemente, a direção do transporte, foi igualmente essencial para o estabelecimento das RA. / Lateral roots (LR) develop from the primary root, whereas adventitious roots (AR) are generally formed from above-ground organs. AR can be formed as an adaptive response to stresses, like wounding or flooding, and their formation is also important for efficient vegetative propagation of economically relevant species, which often depend on clonal propagation of elite genotypes. Hormonal application can stimulate AR development (ARD) and auxins are recognized as major hormones involved in this process. Here, the etiolated seedlings system was used in Arabidopsis thaliana to study several aspects of ARD. Different auxin types, natural or synthetic, were tested and it was found that IAA caused an increase in root number without affecting root length, NAA was effective for ARD, but roots remained short and higher levels of 2,4-D caused callus formation. Through immunolocalization, a higher level of IAA was detected in hypocotyl tissues from which the root primordia differentiated. The expression pattern of genes potentially involved in adventitious rooting was tested by Real-Time PCR. ARD was essentially marked by increased expression of PIN1, SUR2, GH3.3, GH3.6, ARF8 and IAA28. The magnitude of expression of induced genes was much stimulated by NAA, followed by IAA. IAA28 expression increased with ARD, differently from what is known for lateral root development. The auxin receptors TIR1/AFB and ABP1 initiate auxin signaling in the cell through changes in gene expression, selective proteolysis and cell wall loosening. We observed that TIR1/AFB are important in ARD but might be playing redundant roles in the process, whereas ABP1 could be complementing their action. During AR organogenesis, TIR1 and AFB2 seemed to exert greater influence. Auxins are transported in a polar, cell to cell way and depend on several transporters. We analyzed ARD in different mutants affected in auxin influx and efflux transporters, coupled with reporter gene constructs, in presence or absence of exogenous auxin. An essential role was established for AUX1 in AR. Although LAX3 per se was not a key player in the process, it seemed to act in conjunction with AUX1. We also observed that efficient formation of AR depends on the PIN efflux transporters, mainly PIN1, 3 and 7. The proper phosphorylation of PINs by the kinases PID, WAG1 and WAG2, and hence the direction of auxin transport, was equally essential for AR establishment.

Enquanto as raízes laterais (RL) se desenvolvem a partir da raiz primária, as raízes adventícias (RA) são geralmente formadas em órgãos da parte aérea da planta. As RA podem ser formadas como uma resposta adaptativa a estresses, como ferimentos ou alagamentos e a sua formação também é importante para a propagação vegetativa de espécies economicamente relevantes, que frequentemente dependem da propagação clonal de genótipos elite. A aplicação de hormônios pode estimular o desenvolvimento das RA (DRA), e as auxinas são consideradas os principais hormônios envolvidos nesse processo. Neste estudo, o sistema de plântulas estioladas foi usado em Arabidopsis thaliana para analisar diversos aspectos do DRA. Diferentes tipos de auxinas, naturais ou sintéticas, foram testadas e verificou-se que AIA causou um aumento no número de raízes sem afetar seu comprimento, ANA foi efetivo para o DRA, mas as raízes ficaram pequenas, e altas concentrações de 2,4-D causaram a formação de calos. Através de imunolocalização, um nível elevado de AIA foi detectado nos tecidos do hipocótilo que deram origem ao primórdio radicular. O padrão de expressão de genes potencialmente envolvidos com o enraizamento adventício foi testado por PCR em Tempo Real. O DRA foi marcado essencialmente por aumento na expressão de PIN1, SUR2, GH3.3, GH3.6, ARF8 e IAA28. A expressão dos genes induzidos foi mais estimulada por ANA, seguida de AIA. A expressão de IAA28 aumentou com o DRA, diferente do que foi observado no desenvolvimento de RL. Os receptores de auxinas TIR1/AFB e ABP1 iniciam a sinalização de auxinas na célula pelo controle da expressão gênica, proteólise seletiva e afrouxamento da parede celular. Verificou-se que TIR1 e as proteínas AFBs são importantes para o DRA, mas que estes receptores devem estar exercendo funções redundantes no processo e que ABP1 pode agir complementando a sua ação. Durante a organogênese das RA, TIR1 e AFB2 parecem exercer uma maior influência. As auxinas são transportadas de maneira polar, célula a célula e geralmente dependem de transportadores. Analisamos o DRA em diferentes mutantes deficientes no transporte de influxo e efluxo de auxinas juntamente com construções com genes repórteres, na presença ou ausência de auxina exógena. Uma função essencial foi estabelecida para AUX1 no enraizamento adventício e, embora LAX3 per se não tenha sido chave no processo, este parece agir em conjunto com AUX1. Também observamos que a formação eficiente de RA depende dos transportadores de efluxo PIN, principalmente PIN1, 3 e 7. A adequada fosforilação dos PINs pelas quinases PID, WAG1 e WAG2 e, consequentemente, a direção do transporte, foi igualmente essencial para o estabelecimento das RA. / Lateral roots (LR) develop from the primary root, whereas adventitious roots (AR) are generally formed from above-ground organs. AR can be formed as an adaptive response to stresses, like wounding or flooding, and their formation is also important for efficient vegetative propagation of economically relevant species, which often depend on clonal propagation of elite genotypes. Hormonal application can stimulate AR development (ARD) and auxins are recognized as major hormones involved in this process. Here, the etiolated seedlings system was used in Arabidopsis thaliana to study several aspects of ARD. Different auxin types, natural or synthetic, were tested and it was found that IAA caused an increase in root number without affecting root length, NAA was effective for ARD, but roots remained short and higher levels of 2,4-D caused callus formation. Through immunolocalization, a higher level of IAA was detected in hypocotyl tissues from which the root primordia differentiated. The expression pattern of genes potentially involved in adventitious rooting was tested by Real-Time PCR. ARD was essentially marked by increased expression of PIN1, SUR2, GH3.3, GH3.6, ARF8 and IAA28. The magnitude of expression of induced genes was much stimulated by NAA, followed by IAA. IAA28 expression increased with ARD, differently from what is known for lateral root development. The auxin receptors TIR1/AFB and ABP1 initiate auxin signaling in the cell through changes in gene expression, selective proteolysis and cell wall loosening. We observed that TIR1/AFB are important in ARD but might be playing redundant roles in the process, whereas ABP1 could be complementing their action. During AR organogenesis, TIR1 and AFB2 seemed to exert greater influence. Auxins are transported in a polar, cell to cell way and depend on several transporters. We analyzed ARD in different mutants affected in auxin influx and efflux transporters, coupled with reporter gene constructs, in presence or absence of exogenous auxin. An essential role was established for AUX1 in AR. Although LAX3 per se was not a key player in the process, it seemed to act in conjunction with AUX1. We also observed that efficient formation of AR depends on the PIN efflux transporters, mainly PIN1, 3 and 7. The proper phosphorylation of PINs by the kinases PID, WAG1 and WAG2, and hence the direction of auxin transport, was equally essential for AR establishment.

Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...

Cell polarity is a key concept in plant biology. The subcellular localization of Pin- formed (PIN) auxin efflux carriers in the root of "#$%&'()*&* is remarkably asymmetrical, making PINs prominent markers to study cell polarity. In spite of its developmental importance and two decades of research, the molecular basis of PIN polarity remains largely unknown. In this thesis, I employed advanced transgenic and fluorescence microscopy approaches to gain insight into several aspects of PIN polarity regulation. I participated in establishing a novel genetically encoded inhibitor of endocytosis, an invaluable tool for the study of the importance of endocytosis for various cellular processes, including PIN polarity. I demonstrated that apical polarity of PIN2 needs to be re-established after cell division and that this process depends on endocytosis, '+!,(-( protein secretion and the action of WAG1 and related protein kinases, but not transcytosis, cell-cell signaling or intact cytoskeleton. Finally, I identified the previously unknown role of MAB4/MEL proteins in PIN polarity, which lies in the ability of MAB4/MELs to reduce PIN lateral diffusion and thus contribute to PIN polarity maintenance. My results, besides broadening current understanding of PIN polarity regulation, identify mechanisms that...

No description available.