phytosterols and ethylene

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

Topics: Alkaloids; Cyclopentanes; DNA-Binding Proteins; Ethylenes; Gene Expression Regulation, Plant; Oxylipins; Phytosterols; Plant Growth Regulators; Plant Proteins; Plants, Genetically Modified; Promoter Regions, Genetic; Solanum lycopersicum; Species Specificity; Transcription Factors; Transcriptional Activation

Ethylene is an essential hormone in plants that is involved in low-oxygen and reoxygenation responses. As a key transcription factor in ethylene signaling, ETHYLENE INSENSITIVE3 (EIN3) activates targets that trigger various responses. However, most of these targets are still poorly characterized. Through analyses of our microarray data and the published Arabidopsis (Arabidopsis thaliana) EIN3 chromatin immunoprecipitation sequencing data set, we inferred the putative targets of EIN3 during anoxia-reoxygenation. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further studies for its role in tricarboxylic acid cycle replenishment. We demonstrated that both GDH1 and GDH2 are induced during anoxia and reoxygenation and that this induction is mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the level of GDH during anoxia-reoxygenation decreased in the ethylene-insensitive mutants ein2-5 and ein3eil1 Global metabolite analysis indicated that the deamination activity of GDH might regenerate 2-oxoglutarate, which is a cosubstrate that facilitates the breakdown of alanine by alanine aminotransferase when reoxygenation occurs. Moreover, ineffective tricarboxylic acid cycle replenishment, disturbed carbohydrate metabolism, reduced phytosterol biosynthesis, and delayed energy regeneration were found in gdh1gdh2 and ethylene mutants during reoxygenation. Taken together, these data illustrate the essential role of EIN3-regulated GDH activity in metabolic adjustment during anoxia-reoxygenation.

Topics: Anaerobiosis; Arabidopsis; Arabidopsis Proteins; Carbohydrate Metabolism; DNA-Binding Proteins; Energy Metabolism; Ethylenes; Gene Expression Regulation, Plant; Glutamate Dehydrogenase; Metabolome; Models, Biological; Nuclear Proteins; Oxygen; Phenotype; Phytosterols; Protein Stability; Signal Transduction; Transcription Factors

The chilling conditions of apple cold storage can provoke an economically significant necrotic peel disorder called superficial scald (scald) in susceptible cultivars. Disorder development can be reduced by inhibiting ethylene action or oxidative stress as well as intermittent warming. It was previously demonstrated that scald is preceded by a metabolomic shift that results in altered levels of various classes of triterpenoids, including metabolites with mass spectral features similar to β-sitosterol. In this study, a key class of phytosterol metabolites was identified. Changes in peel tissue levels of conjugates of β-sitosterol and campesterol, including acylated steryl glycosides (ASG), steryl glycosides (SG) and steryl esters (SE), as well as free sterols (FS), were determined during the period of scald development. Responses to pre-storage treatment with the ethylene action inhibitor, 1-methylcyclopropene, or an antioxidant (diphenylamine), rapid temperature elevation, and cold acclimation using intermittent warming treatments were evaluated. Diphenylamine, 1-MCP, and intermittent warming all reduced or prevented scald development. ASG levels increased and SE levels decreased in untreated control fruit during storage. Removing fruit from cold storage to ambient temperature induced rapid shifts in ASG and SE fatty acyl moieties from unsaturated to saturated. FS and SG levels remained relatively stable during storage but SG levels increased following a temperature increase after storage. ASG, SE, and SG levels did not increase during 6 months cold storage in fruit subjected to intermittent warming treatment. Overall, the results show that apple peel phytosteryl conjugate metabolism is influenced by storage duration, oxidative stress, ethylene action/ripening, and storage temperature.

Topics: Acclimatization; Antioxidants; Cyclopropanes; Diphenylamine; Ethylenes; Food Preservation; Fruit; Malus; Oxidative Stress; Phytosterols; Plant Diseases; Sitosterols; Temperature

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

Topics: Alkaloids; Base Sequence; Colletotrichum; Escherichia coli; Ethylenes; Fruit; Galactosyltransferases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Silencing; Genes, Plant; Glycosylation; Membrane Proteins; Metabolome; Molecular Sequence Data; Phenotype; Phytosterols; Plant Leaves; Plant Proteins; Plant Roots; Plants, Genetically Modified; Recombinant Proteins; Solanum lycopersicum; Tomatine

Auxin is essential for the formation of the vascular system. We previously reported that a polar auxin transport inhibitor, 1-N-naphthylphthalamic acid (NPA) decreased intracellular auxin levels and prevented tracheary element (TE) differentiation from isolated Zinnia mesophyll cells, but that additional auxin, 1-naphthaleneacetic acid (NAA) overcame this inhibition. To understand the role of auxin in gene regulation during TE differentiation, we performed microarray analysis of genes expressed in NPA-treated cells and NPA-NAA-treated cells. The systematic gene expression analysis revealed that NAA promoted the expression of genes related to auxin signaling and transcription factors that are known to be key regulators of differentiation of procambial and xylem precursor cells. NAA also promoted the expression of genes related to biosynthesis and metabolism of other plant hormones, such as cytokinin, gibberellin and brassinosteroid. Interestingly, detailed analysis showed that NAA rapidly induces the expression of auxin carrier gene homologues. It suggested a positive feedback loop for auxin-regulating vascular differentiation. Based on these results, we discuss the auxin function in early processes of transdifferentiation into TEs.

Topics: Abscisic Acid; Asteraceae; Brassinosteroids; Carrier Proteins; Cell Transdifferentiation; Cells, Cultured; Cholestanols; Cluster Analysis; Cyclopentanes; Cytokinins; Ethylenes; Gene Expression Profiling; Gene Expression Regulation, Plant; Gibberellins; Indoleacetic Acids; Molecular Sequence Data; Naphthaleneacetic Acids; Oligonucleotide Array Sequence Analysis; Oxidoreductases; Oxylipins; Phylogeny; Phytosterols; Plant Growth Regulators; Plant Leaves; Plant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Steroids, Heterocyclic; Xylem

The roles of sterols in plant development are not well understood, but evidence is emerging that they are required for cell division, polarity and patterning by mechanisms that are independent of brassinosteroids, of which they are precursors. Previous evidence shows that two sterol-defective mutants of Arabidopsis thaliana (L.) Heynh., hyd1 and fk(hyd2), are defective in root development. Here we show that the HYD1 gene, like the FK gene, is transcriptionally active in both primary and lateral root meristems, though not in the shoot apical meristem. The patterns of cell division during early stages of lateral root initiation in the hyd1 and fk(hyd2) mutants appear normal. Previous evidence also suggests that auxin and ethylene signalling is defective in the mutants. Here we show that the cytokinin- and ethylene-responsive ACS1::GUS reporter in the fk(hyd2) mutant responds to exogenous cytokinins but not to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicative of normal cytokinin signalling but supporting the hypothesis that ethylene signalling is defective. The defective root meristem cell division activity and expression patterns of the auxin-regulated DR5::GUS and IAA2::GUS reporters can be rescued to a significant extent by the pharmacological or genetic inhibition of ethylene signalling, but not by treatment with aminoethoxyvinylglycine, an inhibitor of ethylene synthesis. This supports the emerging view that the hyd1 and fk(hyd2) mutants exhibit an enhanced and unregulated ethylene signalling activity, which accounts for at least part of the observed mutant phenotypes, including disrupted auxin signalling. The possible relationship between ethylene signalling, membrane sterols and meristem function is discussed.

Topics: Arabidopsis; Cell Division; Ethylenes; Gene Expression Regulation, Plant; Germination; Indoleacetic Acids; Meristem; Mutation; Phytosterols; Plant Roots; Signal Transduction