RESUMEN
Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the nucleotide excision DNA repair system, but this function in plants has not been investigated. We studied the role of MED17 in Arabidopsis plants exposed to UV-B radiation. Our results demonstrate that med17 and OE MED17 plants have altered responses to UV-B, and that MED17 participates in various aspects of the DNA damage response (DDR). Comparison of the med17 transcriptome with that of wild-type (WT) plants showed that almost one-third of transcripts with altered expression in med17 plants were also changed by UV-B exposure in WT plants. Increased sensitivity to DNA damage after UV-B in med17 plants could result from the altered regulation of UV-B responsive transcripts but MED17 also physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we show that MED17 is necessary to regulate the DDR activated by ataxia telangiectasia and Rad3 related (ATR), and that programmed cell death 5 (PDCD5) overexpression reverts the deficiencies in DDR shown in med17 mutants. Our data demonstrate that MED17 is an important regulator of DDR after UV-B irradiation in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Daño del ADN , Reparación del ADN/genética , Rayos UltravioletaRESUMEN
Flavones, one of the largest groups of flavonoids, have beneficial effects on human health and are considered of high nutritional value. Previously, we demonstrated that maize type I flavone synthase (ZmFNSI) is one of the enzymes responsible for the synthesis of O-glycosyl flavones in floral tissues. However, in related species such as rice and sorghum, type II FNS enzymes also contribute to flavone biosynthesis. In this work, we provide evidence that maize has both one FNSI and one FNSII flavone synthases. Arabidopsis transgenic plants expressing each FNS enzyme were generated to validate the role of flavones in protecting plants against UV-B radiation. Here, we demostrate that ZmCYP93G7 (FNSII) has flavone synthase activity and is able to complement the Arabidopsis dmr6 mutant, restoring the susceptibility to Pseudomonas syringae. ZmFNSII expression is controlled by the C1/PL1 + R/B anthocyanin transcriptional complexes, and both ZmFNSI and ZmFNSII are regulated by UV-B. Arabidopsis transgenic plants expressing ZmFNSI or ZmFNSII that accumulate apigenin exhibit less UV-B-induced damage than wild-type plants. Together, we show that maize has two FNS-type enzymes that participate in the synthesis of apigenin, conferring protection against UV-B radiation.
Asunto(s)
Apigenina/fisiología , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas de Plantas/fisiología , Rayos Ultravioleta/efectos adversos , Zea mays/metabolismo , Apigenina/metabolismo , Arabidopsis/metabolismo , Daño del ADN/efectos de la radiación , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Zea mays/enzimología , Zea mays/efectos de la radiaciónRESUMEN
The century-old maize (Zea mays) salmon silks mutation has been linked to the absence of maysin. Maysin is a C-glycosyl flavone that, when present in silks, confers natural resistance to the maize earworm (Helicoverpa zea), which is one of the most damaging pests of maize in America. Previous genetic analyses predicted Pericarp Color1 (P1; R2R3-MYB transcription factor) to be epistatic to the sm mutation. Subsequent studies identified two loci as being capable of conferring salmon silks phenotypes, salmon silks1 (sm1) and sm2 Benefitting from available sm1 and sm2 mapping information and from knowledge of the genes regulated by P1, we describe here the molecular identification of the Sm1 and Sm2 gene products. Sm2 encodes a rhamnosyl transferase (UGT91L1) that uses isoorientin and UDP-rhamnose as substrates and converts them to rhamnosylisoorientin. Sm1 encodes a multidomain UDP-rhamnose synthase (RHS1) that converts UDP-glucose into UDP-l-rhamnose. Here, we demonstrate that RHS1 shows unexpected substrate plasticity in converting the glucose moiety in rhamnosylisoorientin to 4-keto-6-deoxy glucose, resulting in maysin. Both Sm1 and Sm2 are direct targets of P1, as demonstrated by chromatin immunoprecipitation experiments. The molecular characterization of Sm1 and Sm2 described here completes the maysin biosynthetic pathway, providing powerful tools for engineering tolerance to maize earworm in maize and other plants.
Asunto(s)
Flavonoides/biosíntesis , Flavonoides/metabolismo , Glucósidos/biosíntesis , Glucósidos/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/metabolismo , Inmunoprecipitación de Cromatina , Luteolina/metabolismo , Fenotipo , Proteínas de Plantas/genética , Azúcares de Uridina Difosfato/metabolismo , Zea mays/genéticaRESUMEN
DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs.
Asunto(s)
Apoptosis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Reparación del ADN , Raíces de Plantas/fisiología , Plantones/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Daño del ADN , ADN de Plantas/genética , Expresión Génica , Genes Reporteros , Humanos , Mutación , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Hojas de la Planta/efectos de la radiación , Raíces de Plantas/citología , Raíces de Plantas/genética , Raíces de Plantas/efectos de la radiación , Plantas Modificadas Genéticamente , Transporte de Proteínas , Plantones/citología , Plantones/genética , Plantones/efectos de la radiación , Rayos UltravioletaRESUMEN
Flavones are a major group of flavonoids with diverse functions and are extensively distributed in land plants. There are two different classes of FLAVONE SYNTHASE (FNS) enzymes that catalyze the conversion of the flavanones into flavones. The FNSI class comprises soluble Fe(2+)/2-oxoglutarate-dependent dioxygenases, and FNSII enzymes are oxygen- and NADPH-dependent cytochrome P450 membrane-bound monooxygenases. Here, we describe the identification and characterization of FNSI enzymes from maize (Zea mays) and Arabidopsis (Arabidopsis thaliana). In maize, ZmFNSI-1 is expressed at significantly higher levels in silks and pericarps expressing the 3-deoxy flavonoid R2R3-MYB regulator P1, suggesting that ZmFNSI-1 could be the main enzyme for the synthesis of flavone O-glycosides. We also show here that DOWNY MILDEW RESISTANT6 (AtDMR6), the Arabidopsis homologous enzyme to ZmFNSI-1, has FNSI activity. While dmr6 mutants show loss of susceptibility to Pseudomonas syringae, transgenic dmr6 plants expressing ZmFNSI-1 show similar susceptibility to wild-type plants, demonstrating that ZmFNSI-1 can complement the mutant phenotype. AtDMR6 expression analysis showed a tissue- and developmental stage-dependent pattern, with high expression in cauline and senescing leaves. Finally, we show that Arabidopsis cauline and senescing leaves accumulate apigenin, demonstrating that Arabidopsis plants have an FNSI activity involved in the biosynthesis of flavones. The results presented here also suggest cross talk between the flavone and salicylic acid pathways in Arabidopsis; in this way, pathogens would induce flavones to decrease salicylic acid and, hence, increase susceptibility.
Asunto(s)
Arabidopsis/metabolismo , Flavonas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/metabolismo , Apigenina/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Oxigenasas de Función Mixta/genética , Mutación , Filogenia , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Pseudomonas syringae/patogenicidad , Ácido Salicílico/metabolismo , Zea mays/genéticaRESUMEN
Pericarp Color1 (P1) encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize (Zea mays) silks and red phlobaphene pigments in pericarps and other floral tissues, which makes P1 an important visual marker. Using genome-wide expression analyses (RNA sequencing) in pericarps and silks of plants with contrasting P1 alleles combined with chromatin immunoprecipitation coupled with high-throughput sequencing, we show here that the regulatory functions of P1 are much broader than the activation of genes corresponding to enzymes in a branch of flavonoid biosynthesis. P1 modulates the expression of several thousand genes, and â¼1500 of them were identified as putative direct targets of P1. Among them, we identified F2H1, corresponding to a P450 enzyme that converts naringenin into 2-hydroxynaringenin, a key branch point in the P1-controlled pathway and the first step in the formation of insecticidal C-glycosyl flavones. Unexpectedly, the binding of P1 to gene regulatory regions can result in both gene activation and repression. Our results indicate that P1 is the major regulator for a set of genes involved in flavonoid biosynthesis and a minor modulator of the expression of a much larger gene set that includes genes involved in primary metabolism and production of other specialized compounds.
Asunto(s)
Flavonoides/genética , Redes Reguladoras de Genes/genética , Genoma de Planta/genética , Factores de Transcripción/genética , Zea mays/genética , Alelos , Secuencia de Bases , Análisis por Conglomerados , Flavanonas/metabolismo , Flavonoides/análisis , Flavonoides/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Fenotipo , Hojas de la Planta/química , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Propanoles/metabolismo , Semillas/química , Semillas/genética , Semillas/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismo , Activación Transcripcional , Zea mays/química , Zea mays/metabolismoRESUMEN
Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.
Asunto(s)
Glucosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/enzimología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Flavonas/genética , Flavonas/metabolismo , Glucosiltransferasas/genética , Glicósidos/genética , Glicósidos/metabolismo , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Zea mays/genéticaRESUMEN
The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing ProRPL10:ß-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Proteínas Ribosómicas/fisiología , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Prueba de Complementación Genética , Plantas Modificadas Genéticamente/metabolismo , Proteína Ribosómica L10 , Proteínas Ribosómicas/análisis , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Rayos UltravioletaRESUMEN
Flavonols are important compounds for conditional male fertility in maize (Zea mays) and other crops, and they also contribute to protecting plants from UV-B radiation. However, little continues to be known on how maize and other grasses synthesize flavonols, and how flavonol biosynthesis is regulated. By homology with an Arabidopsis flavonol synthase (AtFLS1), we cloned a maize gene encoding a protein (ZmFLS1) capable of converting the dihydrokaempferol (DHK) and dihydroquercetin (DHQ) dihydroflavonols to the corresponding flavonols, kaempferol (K) and quercetin (Q). Moreover, ZmFLS1 partially complements the flavonol deficiency of the Arabidopsis fls1 mutant, and restores anthocyanin accumulation to normal levels. We demonstrate that ZmFLS1 is under the control of the anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. Indeed, using chromatin immunoprecipitation (ChIP) experiments, we establish that ZmFLS1 is an immediate direct target of the P1 and C1/R regulatory complexes, revealing similar control as for earlier steps in the maize flavonoid pathway. Highlighting the importance of flavonols in UV-B protection, we also show that ZmFLS1 is induced in maize seedlings by UV-B, and that this induction is in part mediated by the increased expression of the P1, B and PL1 regulators. Together, our results identify a key flavonoid biosynthetic enzyme so far missed in maize and other monocots, and illustrate mechanisms by which flavonol accumulation is controlled in maize.
Asunto(s)
Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Rayos Ultravioleta , Zea mays/enzimología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Clonación Molecular , Flavonoles/biosíntesis , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Datos de Secuencia Molecular , Oxidorreductasas/genética , Oxidorreductasas/efectos de la radiación , Proteínas de Plantas/genética , Proteínas de Plantas/efectos de la radiación , ARN de Planta/genética , Alineación de Secuencia , Zea mays/genéticaRESUMEN
Ribosomal protein L10 (RPL10) proteins are ubiquitous in the plant kingdom. Arabidopsis (Arabidopsis thaliana) has three RPL10 genes encoding RPL10A to RPL10C proteins, while two genes are present in the maize (Zea mays) genome (rpl10-1 and rpl10-2). Maize and Arabidopsis RPL10s are tissue-specific and developmentally regulated, showing high levels of expression in tissues with active cell division. Coimmunoprecipitation experiments indicate that RPL10s in Arabidopsis associate with translation proteins, demonstrating that it is a component of the 80S ribosome. Previously, ultraviolet-B (UV-B) exposure was shown to increase the expression of a number of maize ribosomal protein genes, including rpl10. In this work, we demonstrate that maize rpl10 genes are induced by UV-B while Arabidopsis RPL10s are differentially regulated by this radiation: RPL10A is not UV-B regulated, RPL10B is down-regulated, while RPL10C is up-regulated by UV-B in all organs studied. Characterization of Arabidopsis T-DNA insertional mutants indicates that RPL10 genes are not functionally equivalent. rpl10A and rpl10B mutant plants show different phenotypes: knockout rpl10A mutants are lethal, rpl10A heterozygous plants are deficient in translation under UV-B conditions, and knockdown homozygous rpl10B mutants show abnormal growth. Based on the results described here, RPL10 genes are not redundant and participate in development and translation under UV-B stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas Ribosómicas/metabolismo , Rayos Ultravioleta , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Técnicas de Silenciamiento del Gen , Familia de Multigenes , Mutagénesis Insercional , Biosíntesis de Proteínas , ARN de Planta/genética , Proteína Ribosómica L10 , Proteínas Ribosómicas/genética , Zea mays/genética , Zea mays/metabolismoRESUMEN
Plant ribosomal proteins play universal roles in translation, although they are also involved in developmental processes and hormone signaling pathways. Among Arabidopsis RPL10 family members, RPL10A exhibits the highest expression during germination and early development, suggesting that RPL10A is the main contributor to these processes. In this work, we first analyzed RPL10A expression pattern in Arabidopsis thaliana using transgenic RPL10Apro:GUS plants. The gene exhibits a ubiquitous expression pattern throughout the plant, but it is most strongly expressed in undifferentiated tissues. Interestingly, gene expression was also detected in stomatal cells. We then examined protein function during seedling establishment and abscisic acid (ABA) response. Heterozygous rpl10A mutant plants show decreased ABA-sensitivity during seed germination, are impaired in early seedling and root development, and exhibit reduced ABA-inhibition of stomatal aperture under light conditions. Overexpression of RPL10A does not affect the germination and seedling growth, but RPL10A-overexpressing lines are more sensitive to ABA during early plant development and exhibit higher stomatal closure under light condition both with and without ABA treatment than wild type plants. Interestingly, RPL10A expression is induced by ABA. Together, we conclude that RPL10A could act as a positive regulator for ABA-dependent responses in Arabidopsis plants.
RESUMEN
In this work, we investigated the role of an Arabidopsis protein, AtPDCD5, during senescence after a 24h-dark period. Previously, we demonstrated that AtPDCD5 participates in programmed cell death (PCD) after UV-B exposure and in age-induced senescence. The results presented here, together with previous data, demonstrate that AtPDCD5 not only plays an important role during DNA damage responses induced by UV-B radiation, but also takes part in PCD programs such as dark-induced senescence in Arabidopsis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Oscuridad , Arabidopsis/genética , Clorofila/metabolismo , Electrólitos/metabolismo , Regulación de la Expresión Génica de las Plantas , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismoRESUMEN
Flavonoids are specialized compounds widely distributed and with diverse functions throughout the plant kingdom and with several benefits for human health. In particular, flavonols, synthesized by flavonol synthase (FLS), protect plants against UV-B radiation and are essential for male fertility in maize and other plants. We have recently characterized a UV-B inducible ZmFLS1, corresponding to the first to be described in monocot plants. Interestingly, the new assembly of the B73 maize genome revealed the presence of a second putative FLS gene (ZmFLS2), with very high identity with ZmFLS1. ZmFLSs expression was analyzed in different maize tissues, and by combining electrophoretic mobility shift assays and transient expression experiments, we show that both genes are direct targets of anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. ZmFLS expression analyses show higher levels of both transcripts in high altitude landraces than inbred lines, and both genes are regulated by UV-B radiation in all lines analyzed. Moreover, the high sequence conservation of the ZmFLS promoters between maize lines suggests that the differences observed in ZmFLS expression are due to allelic variations in the transcription factors that regulate their activities. Finally, we generated pFLS1::FLS1-RFP transgenic plants and analyzed ZmFLS1 expression in different maize tissues; we found that this enzyme is localized in the ER and the perinuclear region.