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Functional analysis of the Arabidopsis PHT4 family of intracellular phosphate transportersGuo, Biwei 15 May 2009 (has links)
The transport of phosphate (Pi) between subcellular compartments is central to
metabolic regulation. Although some of the transporters involved in controlling the
intracellular distribution of Pi have been identified in plants, others are predicted from
genetic and biochemical studies. The Arabidopsis thaliana genome encodes a family of
six proteins that share similarity with SLC17/type I Pi transporters, a diverse group of
animal proteins involved in the transport of Pi, organic anions and chloride.
Heterologous expression in yeast, and gene expression and localization studies in plants
were used to characterize all six members of this Arabidopsis family, which we have
named PHT4. All of the PHT4 proteins mediate Pi transport in yeast with high
specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins
indicate that five of the proteins are targeted to the plastid inner envelope membrane, and
the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and
leaves although two of the genes are expressed predominantly in leaves and one mostly
in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and
chloroplasts, heterotrophic plastids and the Golgi apparatus.
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Functional characterisation of phosphorus uptake pathways in a non-responsive arbuscular mycorrhizal host.Grace, Emily Jane January 2008 (has links)
AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313311 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008
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Functional characterisation of phosphorus uptake pathways in a non-responsive arbuscular mycorrhizal host.Grace, Emily Jane January 2008 (has links)
AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313311 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008
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Phosphorus nutrition of poplarKavka, Mareike Jana 15 December 2016 (has links)
No description available.
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Metabolismo del fosfato en bacterias lácticas y su implicación en la funcionalidadCorrales Benedetti, Daniela 17 January 2025 (has links)
[ES] El metabolismo del fosfato (Pi) es crucial para todos los organismos vivos, tanto en términos energéticos como de biosíntesis. En bacterias lácticas (BAL), como Lacticaseibacillus paracasei, este proceso podría estar vinculado a algunas de sus funciones probióticas, como la retención de metales tóxicos como el arsénico y la síntesis de polifosfato (poli-P), crucial para la homeostasis intestinal. En distintos lactobacilos se han identificado genes (pstSCAB) que codifican un transportador ABC específico de Pi probablemente responsable de acumular arsenato [As(V)], un análogo estructural del Pi. Estos genes se encuentran adyacentes a un sistema de dos componentes (TCS) phoP-phoR, que regula el metabolismo del Pi en otras bacterias. Además, la enzima poli-P quinasa (Ppk) sintetiza poli-P, mientras que su degradación es realizada por la exopolifosfatasa (Ppx). En Lc. paracasei BL23, el gen que codifica Ppk está agrupado con dos genes exopolifosfatasa cuya función no ha sido explorada. El objetivo principal de esta tesis fue estudiar cómo el metabolismo del Pi y su regulación en lactobacilos afectan la captación de As(V) y la producción de poli-P.
Se evaluó el papel del transportador PstSCAB y el TCS PhoPR de lactobacilos en la incorporación de especies inorgánicas de As. Se construyeron mutantes de los genes pstC del transportador PstSCAB, del gen regulador del transporte de Pi phoU y del gen phoP del TCS PhoPR en las cepas Lactiplantibacillus plantarum Lpp+ y WCFS1. Además, se construyeron mutantes en estos genes en nuestra bacteria modelo del laboratorio, Lc. paracasei BL23, incluyendo un mutante en el gen phoR del TCS PhoPR. Se estudió su capacidad de incorporación de As y de crecimiento. Nuestros resultados demostraron que el As(V) probablemente es captado por el sistema de transporte PstSCAB en L. plantarum. También se demostró que la cepa de Lc. paracasei que carece de phoP mostró una mayor resistencia a As(III).
La inesperada resistencia a As(III) en la cepa ¿phoP de Lc. paracasei BL23 motivó la caracterización del regulador transcripcional PhoP. Se realizó un análisis transcriptómico comparativo entre la cepa silvestre y el mutante ¿phoP, mostrando cambios significativos en el transcriptoma. Uno de los hallazgos más destacados fue la disminución en la expresión del gen ackA (acetato quinasa). Esto nos llevó a construir un mutante en este gen, el cual presentó el mismo fenotipo de resistencia a As(III) que la cepa ¿phoP, evidenciando que el déficit en la expresión de la acetato quinasa causante de esta resistencia.
Por otro lado, para explorar los mecanismos implicados en la síntesis y degradación de poli-P en lactobacilos, especialmente las funciones de los genes que codifican las exopolifosfatasas Ppx1 y Ppx2 en Lc. paracasei BL23, se obtuvieron mutantes en sus respectivos genes y se estudió su impacto en la síntesis de poli-P. Se observó un fenotipo inesperado en la falta de Ppx1, que presentó una ausencia total de síntesis de poli-P. Así, Ppx1 es necesaria para la producción in vivo de poli-P. Los análisis catalíticos de Ppk, Ppx1 y Ppx2 y el estudio de las secuencias proteicas sugieren que Ppx1 podría no actuar como exopolifosfatasa. Además, una mutación en el residuo conservado Glu-112 en Ppx1 incrementó la síntesis de poli-P, indicando un papel regulador o catalítico.
Finalmente, se demostró que la transición a la fase estacionaria y la limitación de nutrientes desencadenan la acumulación de poli-P. Se evaluó cómo la variación en los niveles de poli-P afecta la supervivencia bacteriana durante esta fase. Un mutante en ppk2 (otra Ppk en Lc. paracasei BL23) mostró menor supervivencia en fase estacionaria, resaltando su papel en el metabolismo de poli-P.
En conjunto, estos hallazgos profundizan en la comprensión del metabolismo del Pi y del poli-P en Lc. paracasei BL23, con posibles implicaciones en aplicaciones probióticas, especialmente en el contexto de la resistencia a metales tóxicos y la homeostasis intestinal. / [CA] El metabolisme del fosfat (Pi) és fonamental per a tots els éssers vius, tant en termes energètics com de biosíntesi. En bacteris làctics (BAL), com Lacticaseibacillus paracasei, aquest procés està vinculat a diverses funcions probiòtiques, com la retenció de metalls tòxics com l'arsènic i la síntesi de polifosfat (poli-P), essencial per a la homeòstasi intestinal. En diversos lactobacils s'han identificat gens (pstSCAB) que codifiquen un transportador ABC específic de Pi, probablement responsable de l'acumulació d'arsènat [As(V)], un anàleg estructural del Pi. Aquests gens estan adjacents a un sistema de dos components (TCS) phoP-phoR, que regula el metabolisme de Pi en altres bacteris. L'enzim polifosfat quinasa (Ppk) sintetitza poli-P, mentre que la seva degradació es fa per l'exopolifosfatasa (Ppx). En Lc. paracasei BL23, el gen que codifica Ppk està agrupat amb dos gens exopolifosfatasa, la funció dels quals no s'ha explorat fins ara. L'objectiu principal d'aquesta tesi va ser estudiar com el metabolisme de Pi i la seva regulació en lactobacils afecten l'absorció d'As(V) i la producció de poli-P.
Es va avaluar el paper del transportador PstSCAB i del TCS PhoPR en lactobacils en la captació d'espècies inorgàniques d'As. Es van construir mutacions en els gens pstC del transportador PstSCAB, en el gen regulador phoU del transport de Pi i en el gen phoP del TCS PhoPR en les soques Lactiplantibacillus plantarum Lpp+ i WCFS1. També es van construir mutacions en aquests gens en Lc. paracasei BL23, incloent-hi una mutació en el gen phoR del TCS PhoPR. Es va estudiar la capacitat d'incorporació d'As i el creixement de les soques mutants. Els resultats van mostrar que l'As(V) és probablement captat pel sistema de transport PstSCAB en L. plantarum. També es va demostrar que la soca Lc. paracasei sense phoP va presentar major resistència a l'As(III), un resultat inesperat que va motivar la caracterització del regulador transcripcional PhoP.
La resistència a l'As(III) en la soca ¿phoP de Lc. paracasei BL23 va portar a un anàlisi transcriptòmic comparatiu entre la soca salvatge i el mutant ¿phoP. Es va observar una disminució en l'expressió del gen ackA (acetat quinasa), un enzim involucrat en la metabolització d'acetat. Un mutant en aquest gen va mostrar un fenotip de resistència a l'As(III), indicant que el dèficit d'acetat quinasa és responsable de la resistència.
Es va explorar també la síntesi i degradació de poli-P en lactobacils, especialment el paper dels gens que codifiquen les exopolifosfatases Ppx1 i Ppx2 en Lc. paracasei BL23. Es van construir mutants en aquests gens i es va estudiar el seu impacte en la síntesi de poli-P. Es va observar que la manca de Ppx1 impedia la síntesi de poli-P, demostrant que Ppx1 és essencial per a la producció in vivo de poli-P. A més, una mutació en el residu conservat Glu-112 en Ppx1 va augmentar la síntesi de poli-P, suggerint un possible paper regulador o catalític de Ppx1.
Finalment, es va demostrar que la transició a la fase estacionària i la limitació de nutrients desencadenen l'acumulació de poli-P. Un mutant en ppk2 (una altra polifosfat quinasa en Lc. paracasei BL23) va mostrar menor supervivència en la fase estacionària, destacant el paper de Ppk2 en el metabolisme de poli-P.
En resum, aquests resultats contribueixen a una millor comprensió del metabolisme de Pi i poli-P en Lc. paracasei BL23, amb implicacions per a aplicacions probiótiques, especialment en resistència a metalls tòxics i la regulació de la homeòstasi intestinal. / [EN] Phosphate (Pi) metabolism is crucial for all living organisms, both in terms of energy and biosynthesis. In lactic acid bacteria (LAB), such as Lacticaseibacillus paracasei, this process could be linked to some of their probiotic functions, such as the retention of toxic metals like arsenic and the synthesis of polyphosphate (poly-P), which is critical for intestinal homeostasis. In various lactobacilli, genes (pstSCAB) encoding an ABC transporter specific for Pi have been identified. This transporter is likely responsible for accumulating arsenate [As(V)], a structural analog of Pi. These genes are adjacent to a two-component system (TCS) phoP-phoR, which regulates Pi metabolism in other bacteria. Additionally, the enzyme poly-P kinase (Ppk) synthesizes poly-P, while its degradation is carried out by exopolyphosphatase (Ppx). In Lc. paracasei BL23, the gene encoding Ppk is clustered with two exopolyphosphatase genes whose function has not yet been explored. The main objective of this thesis was to study how Pi metabolism and its regulation in lactobacilli affect the uptake of As(V) and poly-P production.
The role of the PstSCAB transporter and the PhoPR TCS in lactobacilli in the incorporation of inorganic As species was evaluated. Mutants were constructed for the pstC genes of the PstSCAB transporter, the phoU gene (regulator of Pi transport), and the phoP gene of the PhoPR TCS in Lactiplantibacillus plantarum strains Lpp+ and WCFS1. Additionally, mutants for these genes were generated in our laboratory model bacterium, Lc. paracasei BL23, including a mutant in the phoR gene of the PhoPR TCS. Their capacity for As uptake and growth was studied. Our results showed that As(V) is likely taken up by the PstSCAB transport system in L. plantarum. It was also demonstrated that the Lc. paracasei strain lacking phoP exhibited greater resistance to As(III).
The unexpected resistance to As(III) in the ¿phoP strain of Lc. paracasei BL23 prompted further characterization of the transcriptional regulator PhoP. A comparative transcriptomic analysis was performed between the wild-type strain and the ¿phoP mutant, revealing significant changes in the transcriptome. One of the most notable findings was the decreased expression of the ackA gene (acetate kinase). This led to the construction of a mutant for this gene, which exhibited the same As(III) resistance phenotype as the ¿phoP strain, demonstrating that reduced acetate kinase expression is the cause of this resistance.
On the other hand, to explore the mechanisms involved in poly-P synthesis and degradation in lactobacilli, particularly the roles of the genes encoding the exopolyphosphatases Ppx1 and Ppx2 in Lc. paracasei BL23, mutants were generated in their respective genes, and their impact on poly-P synthesis was studied. An unexpected phenotype was observed in the absence of Ppx1, which showed a total lack of poly-P synthesis. Thus, Ppx1 is essential for in vivo poly-P production. Catalytic analyses of Ppk, Ppx1, and Ppx2, along with protein sequence studies, suggest that Ppx1 might not act as an exopolyphosphatase. Furthermore, a mutation in the conserved residue Glu-112 in Ppx1 increased poly-P synthesis, indicating a regulatory or catalytic role.
Finally, it was demonstrated that the transition to the stationary phase and nutrient limitation trigger poly-P accumulation. The impact of varying poly-P levels on bacterial survival during this phase was evaluated. A mutant in ppk2 (another Ppk in Lc. paracasei BL23) showed reduced survival in the stationary phase, highlighting its role in poly-P metabolism.
Altogether, these findings enhance the understanding of Pi and poly-P metabolism in Lc. paracasei BL23, with potential implications for probiotic applications, particularly in the context of resistance to toxic metals and intestinal homeostasis. / Corrales Benedetti, D. (2024). Metabolismo del fosfato en bacterias lácticas y su implicación en la funcionalidad [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/214030
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