RESUMO
We investigated the seasonal variation of alkaloids (ephedrine and pseudoephedrine), total polyphenol, and sugar contents in Ephedra sinica cultivated in Japan and elucidated the controlling factors for the variation. In 2018, alkaloids and polyphenol contents increased dramatically from May to July, decreased to their lowest in October, and slightly increased again in November. The reduction of alkaloids and polyphenol contents in the autumn may be affected by precipitation in summer. In 2020, alkaloids and polyphenol contents started to decrease in late July when rainfall was abundant from July to August. In contrast, sucrose and starch contents continued to increase until September and remained high until October. Vascular bundles and fiber developed, and herbal stem weight increased from August to October. Alkaloids and total polyphenol contents tended to increase in November. At the same time, starch and sucrose contents decreased dramatically, whereas glucose and fructose contents increased. Sugar content decreased from October and was lowest in November. The seasonal variation of alkaloids and total polyphenol contents exhibited a contrasting tendency to the seasonal variation of sugar content and tissue development. The seasonal variation of alkaloids and total polyphenol contents was caused by the seasonal variation of sugar content and tissue development. In addition, it is suggested that anatomy may be used for alkaloids content estimation in Ephedra plants.
Assuntos
Alcaloides , Ephedra sinica , Ephedra , Estações do Ano , Japão , Efedrina , SacaroseRESUMO
MADS box transcription factors (TFs) are subdivided into type I and II based on phylogenetic analysis. The type II TFs regulate floral organ identity and flowering time, but type I TFs are relatively less characterized. Here, we report the functional characterization of two type I MADS box TFs in rice (Oryza sativa), MADS78 and MADS79 Transcript abundance of both these genes in developing seed peaked at 48 h after fertilization and was suppressed by 96 h after fertilization, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing MADS78 and MADS 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9-mediated single knockout mutants showed precocious endosperm cellularization. MADS78 and MADS 79 were indispensable for seed development, as a double knockout mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type I MADS box, which enhances nuclear localization. The expression analysis of Fie1, a rice FERTILIZATION-INDEPENDENT SEED-POLYCOMB REPRESSOR COMPLEX2 component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting that Fie1 could be involved in negative regulation of MADS78 and MADS 79 Misregulation of MADS78 and MADS 79 perturbed auxin homeostasis and carbon metabolism, as evident by misregulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show that MADS78 and MADS 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice.
Assuntos
Endosperma/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Domínio MADS/metabolismo , Oryza/crescimento & desenvolvimento , Pólen/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Carbono/metabolismo , Núcleo Celular/metabolismo , Endosperma/genética , Endosperma/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Ácidos Indolacéticos/metabolismo , Proteínas de Domínio MADS/genética , Microscopia Eletrônica de Varredura , Oryza/genética , Oryza/metabolismo , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/metabolismo , Proteínas do Grupo Polycomb/metabolismo , RNA-Seq , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sementes/genética , Sementes/metabolismo , Sementes/ultraestrutura , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Nicotinamide adenine dinucleotide (NAD+ ) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants.
Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , NAD/metabolismo , Antiporters/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Proteínas de Fluorescência Verde , Homeostase , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese Insercional , Proteínas de Transporte de Nucleotídeos , Peroxissomos/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/fisiologia , Amido/metabolismoRESUMO
INTRODUCTION: The native potatoes (Solanum tuberosum ssp. tuberosum L.) cultivated on Chiloé Island in southern Chile have great variability in terms of tuber shape, size, color and flavor. These traits have been preserved throughout generations due to the geographical position of Chiloé, as well as the different uses given by local farmers. OBJECTIVES: The present study aimed to investigate the diversity of metabolites in skin and pulp tissues of eleven native accessions of potatoes from Chile, and evaluate the metabolite associations between tuber tissues. METHODS: For a deeper characterization of these accessions, we performed a comprehensive metabolic study in skin and pulp tissues of tubers, 3 months after harvesting. Specific targeted quantification of metabolites using 96 well microplates, and high-performance liquid chromatography combined with non-targeted metabolite profiling by gas chromatography time-of-flight mass spectrometry were used in this study. RESULTS: We observed differential levels of antioxidant activity and phenolic compounds between skin and pulp compared to a common commercial cultivar (Desireé). In addition, we uncovered considerable metabolite variability between different tuber tissues and between native potatoes. Network correlation analysis revealed different metabolite associations among tuber tissues that indicate distinct associations between primary metabolite and anthocyanin levels, and antioxidant activity in skin and pulp tissues. Moreover, multivariate analysis lead to the grouping of native and commercial cultivars based on metabolites from both skin and pulp tissues. CONCLUSIONS: As well as providing important information to potato producers and breeding programs on the levels of health relevant phytochemicals and other abundant metabolites such as starch, proteins and amino acids, this study highlights the associations of different metabolites in tuber skins and pulp, indicating the need for distinct strategies for metabolic engineering in these tissues. Furthermore, this study shows that native Chilean potato accessions have great potential as a natural source of phytochemicals.
Assuntos
Tubérculos/metabolismo , Solanum tuberosum/classificação , Solanum tuberosum/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Chile , Fenóis/química , Fenóis/metabolismo , Tubérculos/química , Solanum tuberosum/químicaRESUMO
Carbohydrates catabolized via respiratory processes are not only used for energy production but also for biosynthesis of cellular components including soluble molecules (sugars, amino acids, organic acids, and their derivatives) and insoluble macromolecules (proteins, starch, and cell wall). Radiotracer experiments using 14C-labeled glucose provide a global picture of the fate of respired carbon in the metabolic network. This method is based on a chemical fractionation of biomolecules in 14C-glucose fed plant materials and the subsequent determination of radioactivity in each fraction. Metabolic flux into each fraction can be estimated from the specific activity of the hexose phosphate pool. Here, we describe the procedure for glucose metabolism in potato tuber but similar protocols can be adopted for various plant organs and substrates.
Assuntos
Radioisótopos de Carbono/metabolismo , Fracionamento Químico/métodos , Análise do Fluxo Metabólico/métodos , Solanum tuberosum/metabolismo , Respiração Celular , Etanol/química , Glucose/metabolismo , Hexoses/metabolismo , Fosfatos/metabolismo , Solubilidade , Coloração e RotulagemRESUMO
Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , beta-Frutofuranosidase/fisiologia , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Conformação Proteica , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Plântula/crescimento & desenvolvimento , Análise de Sequência de DNA , Vacúolos/enzimologia , Vacúolos/fisiologia , beta-Frutofuranosidase/genéticaRESUMO
Sucrose accumulation in leaves in response to various abiotic stresses suggests a specific role of this disaccharide for stress tolerance and adaptation. The high-affinity transporter StSUT1 undergoes substrate-induced endocytosis presenting the question as to whether altered sucrose accumulation in leaves in response to stresses is also related to enhanced endocytosis or altered activity of the sucrose transporter. StSUT1 is known to interact with several stress-inducible proteins; here we investigated whether one of the interacting candidates, StPDI1, affects its subcellular localization in response to stress: StPDI1 expression is induced by ER-stress and salt. Both proteins, StSUT1 and StPDI1, were found in the detergent resistant membrane (DRM) fraction, and this might affect internalization. Knockdown of StPDI1 expression severely affects abiotic stress tolerance of transgenic potato plants. Analysis of these plants does not reveal modified subcellular localization or endocytosis of StSUT1, but rather a disturbed redox homeostasis, reduced detoxification of reactive oxygen species and effects on primary metabolism. Parallel observations with other StSUT1-interacting proteins are discussed. The redox status in leaves seems to be linked to the sugar status in response to various stress stimuli and to play a role in stress tolerance.
Assuntos
Homeostase , Proteínas de Transporte de Monossacarídeos/fisiologia , Oxirredução , Proteínas de Plantas/fisiologia , Isomerases de Dissulfetos de Proteínas/fisiologia , Sacarose/metabolismo , Clonagem Molecular , Técnicas de Silenciamento de Genes , Homeostase/fisiologia , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Solanum tuberosum/embriologia , Solanum tuberosum/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.
Assuntos
Glucose-1-Fosfato Adenililtransferase/metabolismo , Malatos/metabolismo , Mitocôndrias/metabolismo , Tubérculos/enzimologia , Plastídeos/metabolismo , Solanum tuberosum/enzimologia , Amido/biossíntese , Isótopos de Carbono , Respiração Celular , Fumaratos/metabolismo , Metabolômica , Oxirredução , Plantas Geneticamente Modificadas , Interferência de RNA , Solanum tuberosum/genética , Solanum tuberosum/fisiologiaRESUMO
The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.
Assuntos
Proteínas de Algas/química , Proteínas de Algas/genética , Eucariotos/genética , Células Eucarióticas/fisiologia , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Eucariotos/química , Eucariotos/enzimologia , Dados de Sequência Molecular , Isomerases de Dissulfetos de Proteínas/química , Selênio/fisiologia , Selenoproteínas , Homologia Estrutural de ProteínaRESUMO
We investigated the uptake and bioconcentration of the essential element selenium by a coccolithophorid, Emiliania huxleyi, using [75Se]selenite. The time course of 75Se uptake showed a biphasic pattern, namely a primary phase and a subsequent secondary phase. The primary and secondary phases are due to a rapid selenite uptake process that attained a stationary level within 2 min and a slow Se-accumulation process that continued at a constant rate for 4 h or longer, respectively. Kinetic analysis revealed that the selenite uptake process consists of two components, one saturable and one linearly related to substrate concentration. The Km of the saturable component was 29.8 nM selenite; the uptake activity of this component was suppressed by inhibitors of ATP biogenesis, suggesting that selenite uptake is driven by a high-affinity, active transport system. During a 6-h incubation of cells with [75Se]selenite, 70% of the intracellular 75Se was incorporated into low-molecular-mass compounds (LMCs), and 17% was incorporated into proteins, but [75Se]selenite was barely detectable. A pulse-chase experiment demonstrated that the 75Se that had accumulated in LMCs was transferred into proteins. When the syntheses of amino acids and proteins were each separately inhibited, 75Se incorporation into LMCs and proteins was decreased. These results suggest that E. huxleyi rapidly absorbs selenite, filling a small intracellular pool. Then, Se-containing LMCs are immediately synthesized from the selenite, creating a pool of LMCs that are then metabolized to selenoproteins.