Whole-body insulin resistance and energy expenditure indices, serum lipids, and skeletal muscle metabolome in a state of lipoprotein lipase overexpression.

INTRODUCTION: Overexpression of lipoprotein lipase (LPL) protects against high-fat-diet (HFD)-induced obesity and insulin resistance in transgenic rabbits; however, the molecular mechanisms remain unclear. Skeletal muscle is a major organ responsible for insulin-stimulated glucose uptake and energy expenditure. OBJECTIVES: The main purpose of the current study was to examine the effects of the overexpression of LPL on the skeletal muscle metabolomic profiles to test our hypothesis that the mitochondrial oxidative metabolism would be activated in the skeletal muscle of LPL transgenic rabbits and that the higher mitochondrial oxidative metabolism activity would confer better phenotypic metabolic outcomes. METHODS: Under a HFD, insulin resistance index was measured using the intravenous glucose tolerance test, and total energy expenditure (TEE) was measured by doubly-labeled water in control and LPL transgenic rabbits (n = 12, each group). Serum lipids, such as triglycerides and free fatty acid, were also measured. The skeletal muscle metabolite profile was analyzed using capillary electrophoresis time-of flight mass spectrometry in the two groups (n = 9, each group). A metabolite set enrichment analysis (MSEA) with muscle metabolites and a false discovery rate q < 0.2 was performed to identify significantly different metabolic pathways between the 2 groups. RESULTS: The triglycerides and free fatty acid levels and insulin resistance index were lower, whereas the TEE was higher in the LPL transgenic rabbits than in the control rabbits. Among 165 metabolites detected, the levels of 37 muscle metabolites were significantly different between the 2 groups after false discovery rate correction (q < 0.2). The MSEA revealed that the TCA cycle and proteinogenic amino acid metabolism pathways were significantly different between the 2 groups (P < 0.05). In the MSEA, all four selected metabolites for the TCA cycle (2-oxoglutaric acid, citric acid, malic acid, fumaric acid), as well as eight selected metabolites for proteinogenic amino acid metabolism (asparagine, proline, methionine, phenylalanine, histidine, arginine, leucine, isoleucine) were consistently increased in the transgenic rabbits compared with control rabbits, suggesting that these two metabolic pathways were activated in the transgenic rabbits. Some of the selected metabolites, such as citric acid and methionine, were significantly associated with serum lipids and insulin resistance (P < 0.05). CONCLUSION: The current results suggest that the overexpression of LPL may lead to increased activities of TCA cycle and proteinogenic amino acid metabolism pathways in the skeletal muscle, and these enhancements may play an important role in the biological mechanisms underlying the anti-obesity/anti-diabetes features of LPL overexpression.

An Artificial Conversion of Roots into Organs with Shoot Stem Characteristics by Inducing Two Transcription Factors.

Somatic plant cells can regenerate shoots and/or roots or adventitious embryonic calluses, which may induce organ formation under certain conditions. Such regenerations occur via dedifferentiation of somatic cells, induction of organs, and their subsequent outgrowth. Despite recent advances in understanding of plant regeneration, many details of shoot induction remain unclear. Here, we artificially induced shoot stem-like green organs (SSOs) in Arabidopsis thaliana roots via simultaneous induction of two transcription factors (TFs), ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25, At5g65410) and the B3 family transcription factor REPRODUCTIVE MERISTEM 7 (REM7, At3g18960). The SSOs exhibited negative gravitropism and differentiated vascular bundle phenotypes. The ATHB25/REM7 induced the expression of genes controlling shoot stem characteristics by ectopic expression in roots. Intriguingly, the restoration of root growth was seen in the consecutive and adjacent parts of the SSOs under gene induction conditions. Our findings thus provide insights into the development and regeneration of plant shoot stems.

Metabolic diversification of nitrogen-containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis.

Lysine decarboxylase converts l-lysine to cadaverine as a branching point for the biosynthesis of plant Lys-derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys-derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) and identified cadaverine-derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys-derived alkaloids in the transgenic lines. Moreover, we found several cadaverine-derived metabolites specifically detected in the transgenic lines compared with the non-transformed control. Among these, three specific metabolites were identified and confirmed as 5-aminopentanal, 5-aminopentanoate and δ-valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope-labeled [α-15 N]- or [ε-15 N]-l-lysine. Our results show similar 15 N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys-derived alkaloid biosynthesis.

Precise sequential DNA ligation on a solid substrate: solid-based rapid sequential ligation of multiple DNA molecules.

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.

Effects of molybdenum deficiency and defects in molybdate transporter MOT1 on transcript accumulation and nitrogen/sulphur metabolism in Arabidopsis thaliana.

Molybdenum (Mo) is a micronutrient essential for plant growth, as several key enzymes of plant metabolic pathways contain Mo cofactor in their catalytic centres. Mo-containing oxidoreductases include nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. These are involved in nitrate assimilation, sulphite detoxification, purine metabolism or the synthesis of abscisic acid, auxin and glucosinolates in plants. To understand the effects of Mo deficiency and a mutation in a molybdate transporter, MOT1, on nitrogen and sulphur metabolism in Arabidopsis thaliana, transcript and metabolite profiling of the mutant lacking MOT1 was conducted in the presence or absence of Mo. Transcriptome analysis revealed that Mo deficiency had impacts on genes involved in metabolisms, transport, stress responses, and signal transductions. The transcript level of a nitrate reductase NR1 was highly induced under Mo deficiency in mot1-1. The metabolite profiles were analysed further by using gas chromatography time-of-flight mass spectrometry, capillary electrophoresis time-of-flight mass spectrometry, and ultra high performance liquid chromatography. The levels of amino acids, sugars, organic acids, and purine metabolites were altered significantly in the Mo-deficient plants. These results are the first investigation of the global effect of Mo nutrition and MOT1 on plant gene expressions and metabolism.

An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil.

Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the K(m) value of MOT1 for molybdate is approximately 20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species.

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.