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1.
Nat Commun ; 13(1): 1581, 2022 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-35332155

RESUMEN

Phosphate (Pi) starvation response (PHR) transcription factors play key roles in plant Pi homeostasis maintenance. They are negatively regulated by stand-alone SPX proteins, cellular receptors for inositol pyrophosphate (PP-InsP) nutrient messengers. How PP-InsP-bound SPX interacts with PHRs is poorly understood. Here, we report crystal structures of the rice SPX2/InsP6/PHR2 complex and of the PHR2 DNA binding (MYB) domain in complex with target DNA at resolutions of 3.1 Å and 2.7 Å, respectively. In the SPX2/InsP6/PHR2 complex, the signalling-active SPX2 assembles into a domain-swapped dimer conformation and binds two copies of PHR2, targeting both its coiled-coil (CC) oligomerisation domain and MYB domain. Our results reveal that the SPX2 senses PP-InsPs to inactivate PHR2 by establishing severe steric clashes with the PHR2 MYB domain, preventing DNA binding, and by disrupting oligomerisation of the PHR2 CC domain, attenuating promoter binding. Our findings rationalize how PP-InsPs activate SPX receptor proteins to target PHR family transcription factors.


Asunto(s)
Oryza , Regulación de la Expresión Génica de las Plantas , Homeostasis , Oryza/genética , Oryza/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Factores de Transcripción/metabolismo
2.
Nat Commun ; 12(1): 384, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33452263

RESUMEN

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Difosfatos/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosfatos de Inositol/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/ultraestructura , Cristalografía por Rayos X , Mutación , Proteínas Nucleares/genética , Unión Proteica/genética , Dominios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/ultraestructura
3.
Plant J ; 102(3): 507-516, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31816134

RESUMEN

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Polyphosphates represent important stores of phosphate and energy, and are abundant in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP-binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabidopsis cells by heterologous expression of Escherichia coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme leads to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1-expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron-dense structures in transmission electron microscopy sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalcisomes in the green alga Chlamydomonas reinhardtii.


Asunto(s)
Polifosfatos/metabolismo , Chlamydomonas/metabolismo , Metabolismo Energético/fisiología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo
4.
Elife ; 82019 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-31436531

RESUMEN

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.


Asunto(s)
Arabidopsis/enzimología , Arabidopsis/metabolismo , Difosfatos/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Adenosina Trifosfato/metabolismo , Eliminación de Gen , Homeostasis , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética
5.
PLoS Genet ; 8(2): e1002484, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22346760

RESUMEN

Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.


Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/química , Arabidopsis/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/química , Proteínas Nucleares/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Aminoácidos , Cristalografía por Rayos X , Silenciador del Gen , Datos de Secuencia Molecular , Mutagénesis , Plantas Modificadas Genéticamente , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/genética , Relación Estructura-Actividad
6.
PLoS Genet ; 6(10): e1001175, 2010 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-21060865

RESUMEN

Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved.


Asunto(s)
Arabidopsis/genética , Heterocromatina/genética , Estrés Fisiológico , Transcripción Genética/genética , Arabidopsis/crecimiento & desarrollo , Frío , Perfilación de la Expresión Génica , Glucuronidasa/genética , Glucuronidasa/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilación/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/genética , Plantones/crecimiento & desarrollo , Cloruro de Sodio/farmacología , Temperatura , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos
7.
Plant Cell ; 19(3): 779-90, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17337627

RESUMEN

Animal-mediated pollination is essential in plant reproductive biology and is often associated with pollination syndromes, sets of floral traits, such as color, scent, shape, or nectar content. Selection by pollinators is often considered a key factor in floral evolution and plant speciation. Our aim is the identification and characterization of the genetic changes that caused the evolution of divergent pollination syndromes in closely related plant species. We focus on ANTHOCYANIN2 (AN2), a well-defined myb-type transcription factor that is a major determinant of flower color variation between Petunia integrifolia and Petunia axillaris. Analysis of sequence variation in AN2 in wild P. axillaris accessions showed that loss-of-function alleles arose at least five times independently. DNA sequence analysis was complemented by functional assays for pollinator preference using genetic introgressions and transgenics. These results show that AN2 is a major determinant of pollinator attraction. Therefore, changes in a single gene cause a major shift in pollination biology and support the notion that the adaptation of a flowering plant to a new pollinator type may involve a limited number of genes of large effect. Gene identification and analysis of molecular evolution in combination with behavioral and ecological studies can ultimately unravel the evolutionary genetics of pollination syndromes.


Asunto(s)
Abejas/fisiología , Escarabajos/fisiología , Genes de Plantas , Petunia/genética , Petunia/fisiología , Polen/fisiología , Animales , Teorema de Bayes , Conducta Animal , ADN Complementario/genética , Ecosistema , Evolución Molecular , Datos de Secuencia Molecular , Petunia/clasificación
8.
Genetics ; 168(3): 1585-99, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15579709

RESUMEN

Animal-mediated pollination is essential in the reproductive biology of many flowering plants and tends to be associated with pollination syndromes, sets of floral traits that are adapted to particular groups of pollinators. The complexity and functional convergence of various traits within pollination syndromes are outstanding examples of biological adaptation, raising questions about their mechanisms and origins. In the genus Petunia, complex pollination syndromes are found for nocturnal hawkmoths (P. axillaris) and diurnal bees (P. integrifolia), with characteristic differences in petal color, corolla shape, reproductive organ morphology, nectar quantity, nectar quality, and fragrance. We dissected the Petunia syndromes into their most important phenotypic and genetic components. They appear to include several distinct differences, such as cell-growth and cell-division patterns in the basal third of the petals, elongation of the ventral stamens, nectar secretion and nectar sugar metabolism, and enzymatic differentiation in the phenylpropanoid pathway. In backcross-inbred lines of species-derived chromosome segments in a transposon tagging strain of P. hybrida, one to five quantitative trait loci were identified for each syndrome component. Two loci for stamen elongation and nectar volume were confirmed in introgression lines and showed large allelic differences. The combined data provide a framework for a detailed understanding of floral syndromes from their developmental and molecular basis to their impact on animal behavior. With its molecular genetic tools, this Petunia system provides a novel venue for a pattern of adaptive radiation that is among the most characteristic of flowering plants.


Asunto(s)
Flores/genética , Petunia/genética , Mapeo Cromosómico , Flores/fisiología , Ligamiento Genético , Petunia/fisiología , Fenotipo , Sitios de Carácter Cuantitativo
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