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1.
AMB Express ; 13(1): 98, 2023 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-37735315

RESUMO

Larrea nitida Cav. (Zygophyllaceae) is a plant endemic to Argentina and Chile, and its extract has been studied over the last years due to the presence of antimicrobial agents that can be used to control the growth of some pathogens in agriculture. However, the extract is highly hydrophobic, which strongly affects its fungicidal activity in aqueous media. In this sense, the solid dispersion technique was used to produce L. nitida extract nanodispersions with polyethylene glycol (PLE) and with polyethylene glycol and zinc acetate (PZLE). In order to further evaluate the activity of the extract in PLE and PZLE, blank nanodispersions containing only polyethylene glycol (PEG) and zinc acetate (PZ) without the addition of the extract were also produced. The fungicidal activity of the water-soluble nanoparticles was evaluated at different concentrations (0.037-0.110 g.mL-1). In general, the nanoparticles were successfully produced on a nanometric size and presented a significant inhibitory activity on the growth of the pathogens Fusarium oxysporum and Fusarium verticillioides in aqueous media. Compared to PLE, PZLE presented increased fungistatic activity, possibly due to their increased solubility in water. Even though their application in agriculture should be further investigated, the nanodispersions present great potential to be applied as a green biotechnological tool.

2.
J Exp Bot ; 74(6): 2016-2028, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36575905

RESUMO

Beneficial interactions between plant roots and Trichoderma species lead to both local and systemic enhancements of the plant immune system through a mechanism known as priming of defenses. Previously, we have reported a number of genes and proteins that are differentially regulated in distant tissues of maize plants following inoculation with Trichoderma atroviride. To further investigate the mechanisms involved in the systemic activation of plant responses, here we have further evaluated the regulatory aspects of a selected group of genes when priming is triggered in maize plants. Time-course experiments from the beginning of the interaction between T. atroviride and maize roots followed by leaf infection with Colletotrichum graminicola allowed us to identify a gene set regulated by priming in the leaf tissue. In the same experiment, phytohormone measurements revealed a decrease in jasmonic acid concentration while salicylic acid increased at 2 d and 6 d post-inoculation. In addition, chromatin structure and modification assays showed that chromatin was more open in the primed state compared with unprimed control conditions, and this allowed for quicker gene activation in response to pathogen attack. Overall, the results allowed us to gain insights on the interplay between the phytohormones and epigenetic regulatory events in the systemic and long-lasting regulation of maize plant defenses following Trichoderma inoculation.


Assuntos
Trichoderma , Zea mays , Zea mays/genética , Zea mays/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Ácido Salicílico/metabolismo , Folhas de Planta/metabolismo , Doenças das Plantas/genética , Raízes de Plantas/metabolismo
3.
Planta ; 253(5): 115, 2021 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-33934226

RESUMO

MAIN CONCLUSION: Trichoderma activates plant proteins to counteract Fusarium infection. Comparison between proteomic and transcriptomic data suggests differential response regulation. Proteins from the phenylpropanoid pathway are activated to quickly respond to pathogen attack. Trichoderma species can stimulate local and distant immune responses in colonized plant tissues to prevent future pathogenic attacks. Priming of plant defenses is characterized by changes in transcriptional, metabolic, and epigenetic states after stimulus perception. We have previously investigated transcriptional reprogramming in silk tissues from maize plants inoculated with Trichoderma atroviride and challenged with Fusarium verticillioides (Agostini et al., Mol Plant-Microbe In 32:95-106, 2019). To better understand the molecular changes induced by T. atroviride in maize, a proteomic approach was conducted in this instance. Several proteins belonging to different metabolic categories were detected as priming-involved proteins. However, we detected a very low correlation with those priming-modulated transcripts suggesting the importance of regulatory events a posteriori of the transcriptional process to accomplish the final goal of blocking pathogen entry. Specifically, we focused on the phenylpropanoid pathway, since we detected several proteins that are upregulated in the priming state and might explain cell wall reinforcement as well as the increase in flavonoid and lignin content in maize silks after activation of induced systemic resistance.


Assuntos
Trichoderma , Zea mays , Fusarium , Hypocreales , Doenças das Plantas , Proteoma , Proteômica , Seda
4.
Mol Plant Microbe Interact ; 32(1): 95-106, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30253116

RESUMO

In the present study, we investigated the induced systemic resistance (ISR) activated by the beneficial fungus Trichoderma atroviride in maize plants, and the early immunological responses triggered after challenge with the ear rot pathogen Fusarium verticillioides. By transcriptional analysis, we were able to identify the gene core set specifically modulated in silks of maize plants expressing ISR. Our results showed that the main transcriptional reprogramming falls into genes involved in five main functional categories: cell structure or cell wall, amino acid and protein metabolism, stress responses, signaling, and transport. Among these ISR-related genes, it is important to highlight novel findings regarding hormone metabolism and signaling. The expression of hormone-dependent genes was in good agreement with the abscisic acid, jasmonic acid, and salicylic acid (SA) levels detected in the plants under study. The experimental design allowed the identification of novel regulatory elements related to a heightened state of defense in silks and suggests that steroids and SA are central components of a master regulatory network controlling the immunity of silks during ISR. The results presented also provide evidence about the molecular mechanisms used by maize silks against F. verticillioides to counteract pathogenic development and host invasion, including pathogenesis-related genes, plant cell-wall reinforcement, fungal cell-wall-degrading enzymes and secondary metabolism.


Assuntos
Ácido Salicílico , Transdução de Sinais , Zea mays , Fusarium/imunologia , Regulação da Expressão Gênica de Plantas/imunologia , Ácido Salicílico/imunologia , Transdução de Sinais/imunologia , Zea mays/imunologia , Zea mays/microbiologia
5.
Plant J ; 96(6): 1178-1190, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30238536

RESUMO

Pattern recognition receptors (PRR) and nucleotide-binding leucine-rich repeat proteins (NLR) are major components of the plant immune system responsible for pathogen detection. To date, the transcriptional regulation of PRR/NLR genes is poorly understood. Some PRR/NLR genes are affected by epigenetic changes of neighboring transposable elements (TEs) (cis regulation). We analyzed whether these genes can also respond to changes in the epigenetic marks of distal pericentromeric TEs (trans regulation). We found that Arabidopsis tissues infected with Pseudomonas syringae pv. tomato (Pst) initially induced the expression of pericentromeric TEs, and then repressed it by RNA-directed DNA methylation (RdDM). The latter response was accompanied by the accumulation of small RNAs (sRNAs) mapping to the TEs. Curiously these sRNAs also mapped to distal PRR/NLR genes, which were controlled by RdDM but remained induced in the infected tissues. Then, we used non-infected mom1 (Morpheus' molecule 1) mutants that expressed pericentromeric TEs to test if they lose repression of PRR/NLR genes. mom1 plants activated several PRR/NLR genes that were unlinked to MOM1-targeted TEs, and showed enhanced resistance to Pst. Remarkably, the increased defenses of mom1 were abolished when MOM1/RdDM-mediated pericentromeric TEs silencing was re-established. Therefore, common sRNAs could control PRR/NLR genes and distal pericentromeric TEs and preferentially silence TEs when they are activated.


Assuntos
Arabidopsis/imunologia , Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Genes de Plantas/genética , Imunidade Vegetal/genética , Arabidopsis/genética , Centrômero/genética , Metilação de DNA/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Pseudomonas syringae
6.
Front Plant Sci ; 8: 1179, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28740501

RESUMO

Arabidopsis has 12 histone acetyltransferases grouped in four families: the GNAT/HAG, the MYST/HAM, the p300/CBP/HAC and the TAFII250/HAF families. We previously showed that ham1 and ham2 mutants accumulated higher damaged DNA after UV-B exposure than WT plants. In contrast, hag3 RNA interference transgenic plants showed less DNA damage and lower inhibition of plant growth by UV-B, and increased levels of UV-B-absorbing compounds. These results demonstrated that HAM1, HAM2, and HAG3 participate in UV-B-induced DNA damage repair and signaling. In this work, to further explore the role of histone acetylation in UV-B responses, a putative function of other acetyltransferases of the HAC and the HAF families was analyzed. Neither HAC nor HAF acetyltrasferases participate in DNA damage and repair after UV-B radiation in Arabidopsis. Despite this, haf1 mutants presented lower inhibition of leaf and root growth by UV-B, with altered expression of E2F transcription factors. On the other hand, hac1 plants showed a delay in flowering time after UV-B exposure and changes in FLC and SOC1 expression patterns. Our data indicate that HAC1 and HAF1 have crucial roles for in UV-B signaling, confirming that, directly or indirectly, both enzymes also have a role in UV-B responses.

7.
Front Plant Sci ; 7: 523, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27148340

RESUMO

P1 is a R2R3-MYB transcription factor that regulates the accumulation of a specific group of flavonoids in maize floral tissues, such as flavones and phlobaphenes. P1 is also highly expressed in leaves of maize landraces adapted to high altitudes and higher levels of UV-B radiation. In this work, we analyzed the epigenetic regulation of the P1 gene by UV-B in leaves of different maize landraces. Our results demonstrate that DNA methylation in the P1 proximal promoter, intron1 and intron2 is decreased by UV-B in all lines analyzed; however, the basal DNA methylation levels are lower in the landraces than in B73, a low altitude inbred line. DNA demethylation by UV-B is accompanied by a decrease in H3 methylation at Lys 9 and 27, and by an increase in H3 acetylation. smRNAs complementary to specific regions of the proximal promoter and of intron 2 3' end are also decreased by UV-B; interestingly, P1 smRNA levels are lower in the landraces than in B73 both under control conditions and after UV-B exposure, suggesting that smRNAs regulate P1 expression by UV-B in maize leaves. Finally, we investigated if different P1 targets in flower tissues are also regulated by this transcription factor in response to UV-B. Some targets analyzed show an induction in maize landraces in response to UV-B, with higher basal expression levels in the landraces than in B73; however, not all the transcripts analyzed were found to be regulated by UV-B in leaves.

8.
Plant Cell Environ ; 39(1): 174-84, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26147461

RESUMO

In maize (Zea mays), as well as in other crops, transposable elements (TEs) constitute a great proportion of the genome. Chromatin modifications play a vital role in establishing transposon silencing and perpetuating the acquired repressive state. Nucleosomes associated with TEs are enriched for dimethylation of histone H3 at lysine 9 and 27 (H3K9me2 and H3K27me2, respectively), signals of repressive chromatin. Here, we describe a chromatin protein, ZmMBD101, involved in the regulation of Mutator (Mu) genes in maize. ZmMBD101 is localized to the nucleus and contains a methyl-CpG-binding domain (MBD) and a zinc finger CW (CW) domain. Transgenic lines with reduced levels of ZmMBD101 transcript present enhanced induction of Mu genes when plants are irradiated with UV-B. Chromatin immunoprecipitation analysis with H3K9me2 and H3K27me2 antibodies indicated that ZmMBD101 is required to maintain the levels of these histone repressive marks at Mu terminal inverted repeats (TIRs) under UV-B conditions. Although Mutator inactivity is associated with DNA methylation, cytosine methylation at Mu TIRs is not affected in ZmMBD101 deficient plants. Several plant proteins are predicted to share the simple CW-MBD domain architecture present in ZmMBD101. We hypothesize that plant CW-MBD proteins may also function to protect plant genomes from deleterious transposition.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Genoma de Planta/genética , Proteínas de Plantas/metabolismo , Zea mays/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Núcleo Celular/metabolismo , Cromatina/genética , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Genes Reporter , Histonas/genética , Histonas/metabolismo , Mutação , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão , Raios Ultravioleta , Zea mays/citologia , Zea mays/metabolismo , Zea mays/efeitos da radiação
9.
Plant Signal Behav ; 10(10): e1073871, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26357865

RESUMO

A simple and reproducible method for the treatment of Arabidopsis thaliana leaves with rotenone is presented. Rosette leaves were incubated with rotenone and Triton X-100 for at least 15 h. Treated leaves showed increased expression of COX19 and BCS1a, 2 genes known to be induced in Arabidopsis cell cultures after rotenone treatment. Moreover, rotenone/Triton X-100 incubated leaves presented an inhibition of oxygen uptake. The simplicity of the procedure shows this methodology is useful for studying the effect of the addition of rotenone to a photosynthetic tissue in situ.


Assuntos
Arabidopsis/efeitos dos fármacos , Biologia Celular , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxigênio/metabolismo , Folhas de Planta/efeitos dos fármacos , Rotenona/farmacologia , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Botânica , Técnicas de Cultura de Células , Complexo I de Transporte de Elétrons/metabolismo , Genes de Plantas , Mitocôndrias/metabolismo , Octoxinol , Fotossíntese/efeitos dos fármacos , Folhas de Planta/metabolismo
10.
Front Plant Sci ; 3: 222, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23060891

RESUMO

Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds.

11.
Anal Biochem ; 430(2): 200-2, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22922302

RESUMO

Genome walking procedures are all based on a final polymerase chain reaction amplification, regardless of the strategy employed for the synthesis of the substrate molecule. Here we report a modification of an already established genome walking strategy in which a single-strand DNA substrate is obtained by primer extension driven by Klenow polymerase and which results suitable for the direct sequencing of complex eukaryotic genomes. The efficacy of the method is demonstrated by the identification of nucleotide sequences in the case of two gene families (chiA and P1) in the genomes of several maize species.


Assuntos
Passeio de Cromossomo , DNA Polimerase I/metabolismo , Quitinases/genética , Genoma de Planta , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Zea mays/genética , Zea mays/metabolismo
12.
BMC Plant Biol ; 12: 92, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22702356

RESUMO

BACKGROUND: Plants living at high altitudes are typically exposed to elevated UV-B radiation, and harbor mechanisms to prevent the induced damage, such as the accumulation of UV-absorbing compounds. The maize R2R3-MYB transcription factor P1 controls the accumulation of several UV-B absorbing phenolics by activating a subset of flavonoid biosynthetic genes in leaves of maize landraces adapted to high altitudes. RESULTS: Here, we studied the UV-B regulation of P1 in maize leaves of high altitude landraces, and we investigated how UV-B regulates P1 binding to the CHS promoter in both low and high altitude lines. In addition, we analyzed whether the expansion in the P1 expression domain between these maize landraces and inbred lines is associated to changes in the molecular structure of the proximal promoter, distal enhancer and first intron of P1. Finally, using transient expression experiments in protoplasts from various maize genotypes, we investigated whether the different expression patterns of P1 in the high altitude landraces could be attributed to trans- or cis-acting elements. CONCLUSIONS: Together, our results demonstrate that, although differences in cis-acting elements exist between the different lines under study, the different patterns of P1 expression are largely a consequence of effects in trans.


Assuntos
Altitude , Mutação INDEL , Regiões Promotoras Genéticas , Raios Ultravioleta , Zea mays/efeitos da radiação , Alelos , Sequência de Bases , Imunoprecipitação da Cromatina , Clonagem Molecular , Variações do Número de Cópias de DNA , Regulação da Expressão Gênica de Plantas , Íntrons , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Protoplastos/metabolismo , Protoplastos/efeitos da radiação , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transformação Genética , Zea mays/genética , Zea mays/metabolismo
13.
Plant Physiol Biochem ; 49(6): 646-53, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21398135

RESUMO

Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.


Assuntos
Ananas/enzimologia , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Descarboxilação , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/química , Fotossíntese/fisiologia , Folhas de Planta/química , Proteínas de Plantas/química , Isoformas de Proteínas , Temperatura
14.
Mol Plant ; 4(1): 127-43, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20978083

RESUMO

We characterized the transcriptomic response of transgenic plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase subunit 9. The u-ATP9 transgene driven by A9 and APETALA3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and adenine nucleotides (ATP, ADP) levels without changes in the ATP/ADP ratio. Furthermore, we measured an increase in ROS accumulation and a decrease in glutathione and ascorbate levels with a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by qRT-PCR and enzymatic or functional tests, showed dramatic changes in u-ATP9 plants. Both lines display a modification in the expression of various genes involved in carbon, lipid, and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial respiration, protein synthesis, and degradation are affected. Moreover, the levels of several mRNAs encoding for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone responses, suggesting that several signaling pathways overlap. Indeed, the transcriptome data revealed that the mitochondrial dysfunction dramatically alters the expression of genes involved in signaling pathways, including those related to ethylene, absicic acid, and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this report may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells.


Assuntos
Arabidopsis/metabolismo , Carbono/metabolismo , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Mitocôndrias/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Mitocôndrias/genética , Transdução de Sinais
15.
Biochimie ; 92(7): 909-13, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20359519

RESUMO

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus cells and the pRSETB vector was successful for expression of a soluble His(6)-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.


Assuntos
Arabidopsis/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Triticum/enzimologia , Arabidopsis/genética , Western Blotting , Clonagem Molecular , Eletroforese , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Cinética , Dados de Sequência Molecular , Fosforilação , Triticum/genética
16.
Plant J ; 62(1): 77-91, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20059741

RESUMO

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.


Assuntos
Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Raios Ultravioleta , Zea mays/enzimologia , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Clonagem Molecular , Flavonóis/biossíntese , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Oxirredutases/genética , Oxirredutases/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/efeitos da radiação , RNA de Plantas/genética , Alinhamento de Sequência , Zea mays/genética
17.
Plant Physiol ; 148(3): 1655-67, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18820081

RESUMO

Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants.


Assuntos
Arabidopsis/enzimologia , Citosol/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NAD/metabolismo , Arabidopsis/embriologia , Biocatálise , Gliceraldeído-3-Fosfato Desidrogenases/genética , RNA Mensageiro/genética , Sementes/crescimento & desenvolvimento
18.
Plant Mol Biol ; 61(6): 945-57, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16927206

RESUMO

Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.


Assuntos
Arabidopsis/genética , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/genética , Mutação/genética , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Citosol/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/genética , Microscopia de Fluorescência , Mutagênese Insercional , NADP/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fotossíntese/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxidos/metabolismo
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