RESUMO
The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Carbono/metabolismo , Fosfatos/metabolismo , Dióxido de Carbono/metabolismo , Vitamina B 6 , Piridoxina/metabolismo , Fosfato de Piridoxal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nitrogênio/metabolismoRESUMO
Plant specialized metabolites modulate developmental and ecological functions and comprise many therapeutic and other high-value compounds. However, the mechanisms determining their cell-specific expression remain unknown. Here we describe the transcriptional regulatory network that underlies cell-specific biosynthesis of triterpenes in Arabidopsis thaliana root tips. Expression of thalianol and marneral biosynthesis pathway genes depends on the phytohormone jasmonate and is limited to outer tissues. We show that this is promoted by the activity of redundant bHLH-type transcription factors from two distinct clades and coactivated by homeodomain factors. Conversely, the DOF-type transcription factor DAG1 and other regulators prevent expression of the triterpene pathway genes in inner tissues. We thus show how precise expression of triterpene biosynthesis genes is determined by a robust network of transactivators, coactivators and counteracting repressors.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Triterpenos , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Triterpenos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismoRESUMO
The carbon efficiency of storage lipid biosynthesis from imported sucrose in green Brassicaceae seeds is proposed to be enhanced by the PRK/Rubisco shunt, in which ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts outside the context of the Calvin-Benson-Bassham cycle to recycle CO2 molecules released during fatty acid synthesis. This pathway utilizes metabolites generated by the nonoxidative steps of the pentose phosphate pathway. Photosynthesis provides energy for reactions such as the phosphorylation of ribulose 5-phosphate by phosphoribulokinase (PRK). Here, we show that loss of PRK in Arabidopsis thaliana (Arabidopsis) blocks photoautotrophic growth and is seedling-lethal. However, seeds containing prk embryos develop normally, allowing us to use genetics to assess the importance of the PRK/Rubisco shunt. Compared with nonmutant siblings, prk embryos produce one-third less lipids-a greater reduction than expected from simply blocking the proposed PRK/Rubisco shunt. However, developing prk seeds are also chlorotic and have elevated starch contents compared with their siblings, indicative of secondary effects. Overexpressing PRK did not increase embryo lipid content, but metabolite profiling suggested that Rubisco activity becomes limiting. Overall, our findings show that the PRK/Rubisco shunt is tightly integrated into the carbon metabolism of green Arabidopsis seeds, and that its manipulation affects seed glycolysis, starch metabolism, and photosynthesis.
Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Carbono/metabolismo , Fotossíntese/genética , Sementes/genética , Sementes/metabolismo , Amido/metabolismo , LipídeosRESUMO
[This corrects the article DOI: 10.3389/fpls.2022.903793.].
RESUMO
Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.
RESUMO
Transient transformation methods are frequently used to determine gene function. However, until recently only a few methods have been available in the model medicinal plant Catharanthus roseus. Here, we describe a rapid and highly reproducible protocol for the overexpression of genes of interest by agroinfiltration of C. roseus flower petals. This high throughput method is particularly suitable for screening purposes, for instance, target gene screening of transcription factor candidates, and complements other available methods.
Assuntos
Catharanthus , Catharanthus/genética , Catharanthus/metabolismo , Flores/genética , Flores/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
In plants, primary and specialized metabolism have classically been distinguished as either essential for growth or required for survival in a particular environment. Coenzymes (organic cofactors) are essential for growth but their importance to specialized metabolism is often not considered. In line with the recent proposal of viewing primary and specialized metabolism as an integrated whole rather than segregated lots with a defined interface, we highlight here the importance of collating information on the regulation of coenzyme supply with metabolic demands using examples of vitamin B derived coenzymes. We emphasize that coenzymes can have enormous influence on the outcome of metabolic as well as engineered pathways and should be taken into account in the era of synthetic biology.
Assuntos
Coenzimas , Complexo Vitamínico B , Coenzimas/metabolismo , Plantas/genética , Plantas/metabolismo , Complexo Vitamínico B/metabolismoRESUMO
Stunted growth in saline conditions is a signature phenotype of the Arabidopsis SALT OVERLY SENSITIVE mutants (sos1-5) affected in pathways regulating the salt stress response. One of the mutants isolated, sos4, encodes a kinase that phosphorylates pyridoxal (PL), a B6 vitamer, forming the important coenzyme pyridoxal 5'-phosphate (PLP). Here, we show that sos4-1 and more recently isolated alleles are deficient in phosphorylated B6 vitamers including PLP. This deficit is concomitant with a lowered PL level. Ionomic profiling of plants under standard laboratory conditions (without salt stress) reveals that sos4 mutants are perturbed in mineral nutrient homeostasis, with a hyperaccumulation of transition metal micronutrients particularly in the root, accounting for stress sensitivity. This is coincident with the accumulation of reactive oxygen species, as well as enhanced lignification and suberization of the endodermis, although the Casparian strip is intact and functional. Further, micrografting shows that SOS4 activity in the shoot is necessary for proper root development. Growth under very low light alleviates the impairments, including salt sensitivity, suggesting that SOS4 is important for developmental processes under moderate light intensities. Our study provides a basis for the integration of SOS4 derived B6 vitamers into plant health and fitness.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Organogênese Vegetal/genética , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Fosfato de Piridoxal/genética , Fosfato de Piridoxal/metabolismo , Estresse Salino/genética , Tolerância ao Sal/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Mutação , Raízes de Plantas/genética , Brotos de Planta/genéticaRESUMO
Catharanthus roseus produces a diverse range of specialized metabolites of the monoterpenoid indole alkaloid (MIA) class in a heavily branched pathway. Recent great progress in identification of MIA biosynthesis genes revealed that the different pathway branch genes are expressed in a highly cell type- and organ-specific and stress-dependent manner. This implies a complex control by specific transcription factors (TFs), only partly revealed today. We generated and mined a comprehensive compendium of publicly available C. roseus transcriptome data for MIA pathway branch-specific TFs. Functional analysis was performed through extensive comparative gene expression analysis and profiling of over 40 MIA metabolites in the C. roseus flower petal expression system. We identified additional members of the known BIS and ORCA regulators. Further detailed study of the ORCA TFs suggests subfunctionalization of ORCA paralogs in terms of target gene-specific regulation and synergistic activity with the central jasmonate response regulator MYC2. Moreover, we identified specific amino acid residues within the ORCA DNA-binding domains that contribute to the differential regulation of some MIA pathway branches. Our results advance our understanding of TF paralog specificity for which, despite the common occurrence of closely related paralogs in many species, comparative studies are scarce.
RESUMO
Chloroplasts are essential for autonomous plant growth, and their biogenesis is a complex process requiring both plastid and nuclear genome. One of the essential factors required for chloroplast biogenesis are carotenoids. Carotenoids are synthesized in plastids, and it was shown that plastid localized methylerythritol 4-phosphate (MEP) pathway provides substrates for their biosynthesis. Here, we propose a model, using results of our own mutant analysis combined with the results of others, that a MEP-independent pathway, likely a mevalonate (MVA)-dependent pathway, provides intermediates for chloroplast biogenesis in Arabidopsis embryos. The pattern of this chloroplast biogenesis differs from the MEP-dependent chloroplast biogenesis. In MEP-dependent chloroplast biogenesis, chloroplasts are formed rather uniformly in the whole embryo, with stronger chlorophyll accumulation in cotyledons. In a MEP-independent pathway, chloroplasts are formed predominantly in the hypocotyl and in the embryonic root. We also show that this pattern of chlorophyll accumulation is common to MEP pathway mutants as well as to the mutant lacking geranylgeranyl diphosphate synthase 11 (GGPPS11) activity in plastids but expressing it in the cytosol (GGPPS11cyt). It was recently described that shorter GGPPS11 transcripts are present in Arabidopsis, and they can be translated into active cytosolic proteins. We therefore propose that the MEP-independent pathway for chloroplast biogenesis in Arabidopsis embryos is an MVA pathway that provides substrates for the synthesis of GGPP via GGPPS11cyt and this is then transported to plastids, where it is used for carotenoid biosynthesis and subsequently for chloroplast biogenesis mainly in the hypocotyl and in the embryonic root.
RESUMO
Phytohormones regulate the plasticity of plant growth and development, and responses to biotic and abiotic stresses. Many hormone signal transduction cascades involve ubiquitination and subsequent degradation of proteins by the 26S proteasome. The conjugation of ubiquitin to a substrate is facilitated by the E1 activating, E2 conjugating, and the substrate-specifying E3 ligating enzymes. The most prevalent type of E3 ligase in plants is the Cullin-RING ligase (CRL)-type, with F-box proteins (FBPs) as the substrate recognition component. The activity of these SKP-Cullin-F-box (SCF) complexes needs to be tightly regulated in time and place. Here, we review the regulation of SCF function in plants on multiple levels, with a focus on the auxin and jasmonate SCF-type receptor complexes. We discuss in particular the relevance of protein-protein interactions and post-translational modifications as mechanisms to keep SCF functioning under control. Additionally, we highlight the unique property of SCFTIR1/AFB and SCFCOI1 to recognize substrates by forming co-receptor complexes. Finally, we explore how engineered selective agonists can be used to study and uncouple the outcomes of the complex auxin and jasmonate signaling networks that are governed by these FBPs.
Assuntos
Ciclopentanos/metabolismo , Proteínas F-Box , Ácidos Indolacéticos/metabolismo , Oxilipinas/metabolismo , Proteínas Ligases SKP Culina F-Box , Viridiplantae/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Genes de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Ubiquitinação , Viridiplantae/crescimento & desenvolvimentoRESUMO
Metabolic pathways are tightly regulated at the transcriptional and post-translational level, often relying on protein-protein interactions or post-translational protein modifications. Whereas these principles have been established already for a long time, the number of experimentally established cases is expected to rise exponentially in the near future as a result of recent advances in protein-based detection methods. Interactions and modifications are often dependent on only short amino-acid sequences that represent excellent targets for new gene editing technologies by which specific base pairs can be exchanged. Here, we introduce the concept of metabolic editing, which is based on identifying specific amino-acid sequences that are subsequently targeted for gene editing. The proposed workflow will serve for both applied metabolic engineering purposes and proof-of-concept studies in fundamental research.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Engenharia Metabólica , Processamento de Proteína Pós-TraducionalRESUMO
To fend off microbial pathogens and herbivores, plants have evolved a wide range of defense strategies such as physical barriers, or the production of anti-digestive proteins or bioactive specialized metabolites. Accumulation of the latter compounds is often regulated by transcriptional activation of the biosynthesis pathway genes by the phytohormone jasmonate-isoleucine. Here, we used our recently developed flower petal transformation method in the medicinal plant Catharanthus roseus to shed light on the complex regulatory mechanisms steering the jasmonate-modulated biosynthesis of monoterpenoid indole alkaloids (MIAs), to which the anti-cancer compounds vinblastine and vincristine belong. By combinatorial overexpression of the transcriptional activators BIS1, ORCA3 and MYC2a, we provide an unprecedented insight into the modular transcriptional control of MIA biosynthesis. Furthermore, we show that the expression of an engineered de-repressed MYC2a triggers a tremendous reprogramming of the MIA pathway, finally leading to massively increased accumulation of at least 23 MIAs. The current study unveils an innovative approach for future metabolic engineering efforts for the production of valuable bioactive plant compounds in non-model plants.
Assuntos
Apocynaceae , Engenharia Metabólica , Proteínas de Plantas , Plantas Geneticamente Modificadas , Alcaloides de Triptamina e Secologanina/metabolismo , Fatores de Transcrição , Apocynaceae/genética , Apocynaceae/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plants produce countless specialized compounds of diverse chemical nature and biological activities. Their biosynthesis often exclusively occurs either in response to environmental stresses or is limited to dedicated anatomical structures. In both scenarios, regulation of biosynthesis appears to be mainly controlled at the transcriptional level, which is generally dependent on a combined interplay of DNA-related mechanisms and the activity of transcription factors that may act in a combinatorial manner. How environmental and developmental cues are integrated into a coordinated cell type-specific stress response has only partially been unraveled so far. Building on the available examples from (metabolic) gene expression, here we propose theoretical models of how this integration of signals may occur at the level of transcriptional control.
Assuntos
Regulação da Expressão Gênica de Plantas , Plantas/metabolismo , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Redes e Vias Metabólicas , Oxilipinas/metabolismo , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/fisiologiaRESUMO
Monoterpenoid indole alkaloids (MIAs) are plant defense compounds and high-value pharmaceuticals. Biosynthesis of the universal MIA precursor, secologanin, is organized between internal phloem-associated parenchyma (IPAP) and epidermis cells. Transporters for intercellular transport of proposed mobile pathway intermediates have remained elusive. Screening of an Arabidopsis thaliana transporter library expressed in Xenopus oocytes identified AtNPF2.9 as a putative iridoid glucoside importer. Eight orthologs were identified in Catharanthus roseus, of which three, CrNPF2.4, CrNPF2.5 and CrNPF2.6, were capable of transporting the iridoid glucosides 7-deoxyloganic acid, loganic acid, loganin and secologanin into oocytes. Based on enzyme expression data and transporter specificity, we propose that several enzymes of the biosynthetic pathway are present in both IPAP and epidermis cells, and that the three transporters are responsible for transporting not only loganic acid, as previously proposed, but multiple intermediates. Identification of the iridoid glucoside-transporting CrNPFs is an important step toward understanding the complex orchestration of the seco-iridioid pathway.
Assuntos
Catharanthus/metabolismo , Glucosídeos Iridoides/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Animais , Bioensaio , Transporte Biológico , Vias Biossintéticas/genética , Catharanthus/genética , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Iridoides/metabolismo , Cinética , Modelos Biológicos , Oócitos/metabolismo , Transporte Proteico , Terpenos/metabolismo , Xenopus/metabolismoRESUMO
The essential micronutrient vitamin B6 is best known in its enzymatic cofactor form, pyridoxal 5'-phosphate (PLP). However, vitamin B6 comprises the amine pyridoxamine 5'-phosphate (PMP) and the alcohol pyridoxine 5'-phosphate (PNP) in addition to PLP, as well as their corresponding non-phosphorylated forms. The different B6 forms (called vitamers) are enzymatically interconverted in a ubiquitous salvage pathway. Recently, we have shown that balancing the ratio of the different B6 vitamers in particular PMP by the PMP/PNP oxidase PDX3 is essential for growth and development in Arabidopsis thaliana. Intriguingly, nitrate to ammonium conversion is impaired in pdx3 mutants, such that the mutants become ammonium-dependent, suggesting an interaction between vitamin B6 and nitrogen metabolism. In addition, we found a strong up-regulation of genes related to plant defense. Here, we further show that pdx3 mutants display a temperature-sensitive phenotype that is typical of autoimmune mutants and is possibly connected to the impaired nitrogen metabolism.
Assuntos
Autoimunidade , Nitrogênio/metabolismo , Vitamina B 6/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Fenótipo , Reprodução , TemperaturaRESUMO
Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5'-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 5'-phosphate (PMP), pyridoxine 5'-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B6 homeostasis and nitrogen metabolism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitrogênio/metabolismo , Fosfato de Piridoxal/análogos & derivados , Piridoxamina/análogos & derivados , Piridoxaminafosfato Oxidase/metabolismo , Vitamina B 6/metabolismo , Compostos de Amônio/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Vias Biossintéticas , Homeostase , Metaboloma , Modelos Biológicos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Piridoxamina/química , Piridoxamina/metabolismo , Piridoxaminafosfato Oxidase/genética , Reprodução , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Análise de Sequência de RNA , Vitamina B 6/químicaRESUMO
Most plastid isoprenoids, including photosynthesis-related metabolites such as carotenoids and the side chain of chlorophylls, tocopherols (vitamin E), phylloquinones (vitamin K), and plastoquinones, derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. Seven out of 10 functional GGPPS isozymes in Arabidopsis thaliana reside in plastids. We aimed to address the function of different GGPPS paralogues for plastid isoprenoid biosynthesis. We constructed a gene co-expression network (GCN) using GGPPS paralogues as guide genes and genes from the upstream and downstream pathways as query genes. Furthermore, knock-out and/or knock-down ggpps mutants were generated and their growth and metabolic phenotypes were analyzed. Also, interacting protein partners of GGPPS11 were searched for. Our data showed that GGPPS11, encoding the only plastid isozyme essential for plant development, functions as a hub gene among GGPPS paralogues and is required for the production of all major groups of plastid isoprenoids. Furthermore, we showed that the GGPPS11 protein physically interacts with enzymes that use GGPP for the production of carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. GGPPS11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. Both gene co-expression and protein-protein interaction likely contribute to the channeling of GGPP by GGPPS11.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Terpenos/metabolismo , Alquil e Aril Transferases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carotenoides/metabolismo , Clorofila/metabolismo , Isoenzimas , Fenótipo , Fotossíntese , Plastídeos/enzimologia , Fosfatos de Poli-Isoprenil/metabolismo , Mapeamento de Interação de ProteínasRESUMO
Plants use B vitamin compounds as cofactors for metabolism. Biosynthesis de novo of these metabolites in plants is almost fully elucidated. However, salvaging of precursors as well as cofactor derivatives is only being unraveled. Furthermore, processing of these compounds when damaged by cellular activities to prevent deleterious effects on metabolism is emerging. Recent investigations indicate that the role of B vitamins goes beyond metabolism and are being linked with epigenetic traits, specific developmental cues, the circadian clock, as well as abiotic and biotic stress responses. More in depth investigations on the regulation of the provision of these compounds through biosynthesis de novo, salvage and transport is suggesting that plants may share the cost of this load by division of labor.
Assuntos
Desenvolvimento Vegetal , Plantas/metabolismo , Vitaminas/metabolismo , Epigenômica , Regulação da Expressão Gênica de Plantas , Fenômenos Fisiológicos Vegetais , Plantas/genética , Estresse FisiológicoRESUMO
Unwanted enzyme side reactions and spontaneous decomposition of metabolites can lead to a build-up of compounds that compete with natural enzyme substrates and must be dealt with for efficient metabolism. It has recently been realized that there are enzymes that process such compounds, formulating the concept of metabolite repair. NADH and NADPH are vital cellular redox cofactors but can form non-functional hydrates (named NAD(P)HX) spontaneously or enzymatically that compete with enzymes dependent on NAD(P)H, impairing normal enzyme function. Here we report on the functional characterization of components of a potential NAD(P)H repair pathway in plants comprising a stereospecific dehydratase (NNRD) and an epimerase (NNRE), the latter being fused to a vitamin B6 salvage enzyme. Through the use of the recombinant proteins, we show that the ATP-dependent NNRD and NNRE act concomitantly to restore NAD(P)HX to NAD(P)H. NNRD behaves as a tetramer and NNRE as a dimer, but the proteins do not physically interact. In vivo fluorescence analysis demonstrates that the proteins are localized to mitochondria and/or plastids, implicating these as the key organelles where this repair is required. Expression analysis indicates that whereas NNRE is present ubiquitously, NNRD is restricted to seeds but appears to be dispensable during the normal Arabidopsis life cycle.