Trehalose 6–phosphate coordinates organic and amino acid metabolism with carbon availability

Trehalose 6–phosphate (Tre6P) is an essential signal metabolite in plants, linking growth and development to carbon metabolism. The sucrose–Tre6P nexus model postulates that Tre6P acts as both a signal and negative feedback regulator of sucrose levels. To test this model, short‐term metabolic responses to induced increases in Tre6P levels were investigated in Arabidopsis thaliana plants expressing the Escherichia coli Tre6P synthase gene (otsA) under the control of an ethanol‐inducible promoter. Increased Tre6P levels led to a transient decrease in sucrose content, post‐translational activation of nitrate reductase and phosphoenolpyruvate carboxylase, and increased levels of organic and amino acids. Radio‐isotope (¹⁴CO₂) and stable isotope (¹³CO₂) labelling experiments showed no change in the rates of photoassimilate export in plants with elevated Tre6P, but increased labelling of organic acids. We conclude that high Tre6P levels decrease sucrose levels by stimulating nitrate assimilation and anaplerotic synthesis of organic acids, thereby diverting photoassimilates away from sucrose to generate carbon skeletons and fixed nitrogen for amino acid synthesis. These results are consistent with the sucrose–Tre6P nexus model, and implicate Tre6P in coordinating carbon and nitrogen metabolism in plants.     

Trehalose is required for growth of Mycobacterium smegmatis

Mycobacteria contain high levels of the disaccharide trehalose in free form as well as within various immunologically relevant glycolipids such as cord factor and sulfolipid-1. By contrast, most bacteria use trehalose solely as a general osmoprotectant or thermoprotectant. Mycobacterium tuberculosis and Mycobacterium smegmatis possess three pathways for the synthesis of trehalose. Most bacteria possess only one trehalose biosynthesis pathway and do not elaborate the disaccharide into more complex metabolites, suggesting a distinct role for trehalose in mycobacteria. We disabled key enzymes required for each of the three pathways in M. smegmatis by allelic replacement. The resulting trehalose biosynthesis mutant was unable to proliferate and enter stationary phase unless supplemented with trehalose. At elevated temperatures, however, the mutant was unable to proliferate even in the presence of trehalose. Genetic complementation experiments showed that each of the three pathways was able to recover the mutant in the absence of trehalose, even at elevated temperatures. From a panel of trehalose analogs, only those with the native alpha,alpha-(1,1) anomeric stereochemistry rescued the mutant, whereas alternate stereoisomers and general osmo- and thermoprotectants were inactive. These findings suggest a dual role for trehalose as both a thermoprotectant and a precursor of critical cell wall metabolites.     

Trehalose mediated growth inhibition of Arabidopsis seedlings is due to trehalose-6-phosphate accumulation

Trehalose-6-phosphate (T6P) is required for carbon utilization during Arabidopsis development, and its absence is embryo lethal. Here we show that T6P accumulation inhibits seedling growth. Wild-type seedlings grown on 100 mm trehalose rapidly accumulate T6P and stop growing, but seedlings expressing Escherichia coli trehalose phosphate hydrolase develop normally on such medium. T6P accumulation likely results from much-reduced T6P dephosphorylation when trehalose levels are high. Metabolizable sugars added to trehalose medium rescue T6P inhibition of growth. In addition, Suc feeding leads to a progressive increase in T6P concentrations, suggesting that T6P control over carbon utilization is related to available carbon for growth. Expression analysis of genes from the Arabidopsis trehalose metabolism further supports this: Suc rapidly induces expression of trehalose phosphate synthase homolog AtTPS5 to high levels. In contrast, T6P accumulation after feeding trehalose in the absence of available carbon induces repression of genes encoding T6P synthases and expression of T6P phosphatases. To identify processes controlled by T6P, we clustered expression profile data from seedlings with altered T6P content. T6P levels correlate with expression of a specific set of genes, including the S6 ribosomal kinase ATPK19, independently of carbon status. Interestingly, Suc addition represses 15 of these genes, one of which is AtKIN11, encoding a Sucrose Non Fermenting 1 (SNF1)-related kinase known to play a role in Suc utilization.     

HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis

Post-translational modifications of histones, including acetylation, play a key role in modulating dynamic changes in chromatin structure and gene activity. Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases. HDA6 is a RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering (HDA6-RNAi) plants displayed higher levels of acetylated H3 compared with wild-type, suggesting that HDA6 affects histone acetylation levels globally. The expression of the jasmonate responsive genes, PDF1.2, VSP2, JIN1, and ERF1, was down-regulated in axe1-5 and HDA6-RNAi plants. Furthermore, axe1-5 and HDA6-RNAi plants displayed increased leaf longevity compared with the wild type. The expression of the senescence-associated genes, SAG12 and SEN4, was down-regulated in the axe1-5 and HDA6-RNAi plants. In addition, axe1-5 and HDA6-RNAi plants displayed late-flowering. The expression of FLC was up-regulated and hyperacetylated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and thereby repress its expression. Our results suggest that HDA6 is involved in jasmonate response, senescence, and flowering in Arabidopsis.     

Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.     

Premature leaf senescence 3, encoding a methyltransferase,is required for melatonin biosynthesis in rice

Premature leaf senescence in rice is one of the most common factors affecting the plant's development and yield. Although methyltransferases are involved in diverse biological functions, their roles in rice leaf senescence have not been previously reported. In this study, we identified the premature leaf senescence 3 (pls3) mutant in rice, which led to early leaf senescence and early heading date. Further investigations revealed that premature leaf senescence was triggered by the accumulation of reactive oxygen species. Using physiological analysis, we found that chlorophyll content was reduced in the pls3 mutant leaves, while hydrogen peroxide (H₂O₂) and malondialdehyde levels were elevated. Consistent with these findings, the pls3 mutant exhibited hypersensitivity to exogenous hydrogen peroxide. The expression of other senescence‐associated genes such as Osh36 and RCCR1 was increased in the pls3 mutant. Positional cloning indicated the pls3 phenotype was the result of a mutation in OsMTS1, which encodes an O‐methyltransferase in the melatonin biosynthetic pathway. Functional complementation of OsMTS1 in pls3 completely restored the wild‐type phenotype. We found leaf melatonin content to be dramatically reduced in pls3, and that exogenous application of melatonin recovered the pls3 mutant's leaf senescence phenotype to levels comparable to that of wild‐type rice. Moreover, overexpression of OsMTS1 in the wild‐type plant increased the grain yield by 15.9%. Our results demonstrate that disruption of OsMTS1, which codes for a methyltransferase, can trigger leaf senescence as a result of decreased melatonin production.     

Osteopontin is required for the early onset of high fat diet-induced insulin resistance in mice

Background

Insulin resistance is manifested in muscle, adipose tissue, and liver and is associated with adipose tissue inflammation. The cellular components and mechanisms that regulate the onset of diet-induced insulin resistance are not clearly defined.

Methodology and Principal Findings

We initially observed osteopontin (OPN) mRNA over-expression in adipose tissue of obese, insulin resistant humans and rats which was normalized by thiazolidinedione (TZD) treatment in both species. OPN regulates inflammation and is implicated in pathogenic maladies resulting from chronic obesity. Thus, we tested the hypothesis that OPN is involved in the early development of insulin resistance using a 2–4 week high fat diet (HFD) model. OPN KO mice fed HFD for 2 weeks were completely protected from the severe skeletal muscle, liver and adipose tissue insulin resistance that developed in wild type (WT) controls, as determined by hyperinsulinemic euglycemic clamp and acute insulin-stimulation studies. Although two-week HFD did not alter body weight or plasma free fatty acids and cytokines in either strain, HFD-induced hyperleptinemia, increased adipose tissue inflammation (macrophages and cytokines), and adipocyte hypertrophy were significant in WT mice and blunted or absent in OPN KO mice. Adipose tissue OPN protein isoform expression was significantly altered in 2- and 4-week HFD-fed WT mice but total OPN protein was unchanged. OPN KO bone marrow stromal cells were more osteogenic and less adipogenic than WT cells in vitro. Interestingly, the two differentiation pathways were inversely affected by HFD in WT cells in vitro.

Conclusions

The OPN KO phenotypes we report reflect protection from insulin resistance that is associated with changes in adipocyte biology and adipose tissue inflammatory status. OPN is a key component in the development of HFD-induced insulin resistance.     

Rising rates of starch degradation during daytime and trehalose 6-phosphate optimize carbon availability

Many plants, including Arabidopsis (Arabidopsis thaliana), accumulate starch in the light and remobilize it to support maintenance and growth at night. Starch synthesis and degradation are usually viewed as temporally separate processes. Recently, we reported that starch is also degraded in the light. Degradation rates are generally low early in the day but rise with time. Here, we show that the rate of degradation in the light depends on time relative to dawn rather than dusk. We also show that degradation in the light is inhibited by trehalose 6-phosphate, a signal for sucrose availability. The observed responses of degradation in the light can be simulated by a skeletal model in which the rate of degradation is a function of starch content divided by time remaining until dawn. The fit is improved by extension to include feedback inhibition of starch degradation by trehalose 6-phosphate. We also investigate possible functions of simultaneous starch synthesis and degradation in the light, using empirically parameterized models and experimental approaches. The idea that this cycle buffers growth against falling rates of photosynthesis at twilight is supported by data showing that rates of protein and cell wall synthesis remain high during a simulated dusk twilight. Degradation of starch in the light may also counter over-accumulation of starch in long photoperiods and stabilize signaling around dusk. We conclude that starch degradation in the light is regulated by mechanisms similar to those that operate at night and is important for stabilizing carbon availability and signaling, thus optimizing growth in natural light conditions.

Starch degradation in the light is regulated by similar mechanisms to those operating at night, stabilizing carbon availability, and thereby optimizing growth in natural light conditions     

Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence

The onset of leaf senescence is controlled by leaf age and ethylene can promote leaf senescence within a specific age window. We exploited the interaction between leaf age and ethylene and isolated mutants with altered leaf senescence that are named as onset of leaf death (old) mutants. Early leaf senescence mutants representing three genetic loci were selected and their senescence syndromes were characterised using phenotypical, physiological and molecular markers. old1 is represented by three recessive alleles and displayed earlier senescence both in air and upon ethylene exposure. The etiolated old1 seedlings exhibited a hypersensitive triple response. old2 is a dominant trait and the mutant plants were indistinguishable from the wild-type when grown in air but showed an earlier senescence syndrome upon ethylene treatment. old3 is a semi-dominant trait and its earlier onset of senescence is independent of ethylene treatment. Analyses of the chlorophyll degradation, ion leakage and SAG expression showed that leaf senescence was advanced in ethylene-treated old2 plants and in both air-grown and ethylene-treated old1 and old3 plants. Epistatic analysis indicated that OLD1 might act downstream of OLD2 and upstream of OLD3 and mediate the interaction between leaf age and ethylene. A genetic model was proposed that links the three OLD genes and ethylene into a regulatory pathway controlling the onset of leaf senescence.     

Autophagy is required for lipid homeostasis during dark-induced senescence

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon-starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) subjected to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming toward lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants.

Autophagy is required for the mobilization of membrane lipid components and lipid droplet dynamics during extended darkness in Arabidopsis.     

Fructose-6-phosphate is not a substrate for glucose-6-phosphate dehydrogenase

D-Fructose-6-phosphate was shown not to be a substrate for glucose-6-phosphate dehydrogenases (EC. 1.1.1.49) from human erythrocytes, bovine adrenal, rat liver, three yeasts (brewer's yeast, baker's yeast, and Candida utilis), and Leuconostoc mesenteroides. These findings contrast with those of G.M. Kidder (J. Exp. Zool., 226:385-390, '83).     

Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co²⁺ and Mg²⁺ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.     

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.     

Glucose-6-phosphate dehydrogenase-deficient cells show an increased propensity for oxidant-induced senescence

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and the maintenance of cellular redox balance. We previously showed that G6PD-deficient fibroblasts undergo growth retardation and premature cellular senescence. In the present study, we demonstrate abatement of both the intracellular G6PD activity and the ratio NADPH/NADP(+) during the serial passage of G6PD-deficient cells. This was accompanied by a significant increase in the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). This suggests that the lowered resistance to oxidative stress and accumulative oxidative damage may account for the premature senescence of these cells. Consistent with this, the G6PD-deficient cells had an increased propensity for hydrogen peroxide (H(2)O(2))-induced senescence; these cells exhibited such senescent phenotypes as large, flattened morphology and increased senescence-associated beta-galactosidase (SA-beta-Gal) staining. Decreases in both the intracellular G6PD activity and the NADPH/NADP(+) ratio were concomitant with an increase in 8-OHdG level in H(2)O(2)-induced senescent cells. Exogenous expression of G6PD protected the deficient cells from stress-induced senescence. No significant telomere shortening occurred upon repetitive treatment with H(2)O(2). Simultaneous induction of p16(INK4a) and p53 was detected in G6PD-deficient but not in normal fibroblasts during H(2)O(2)-induced senescence. Our findings support the notion that G6PD status, and thus proper redox balance, is a determinant of cellular senescence.     

Genome-wide evaluation of histone methylation changes associated with leaf senescence in Arabidopsis

Leaf senescence is the orderly dismantling of older tissue that allows recycling of nutrients to developing portions of the plant and is accompanied by major changes in gene expression. Histone modifications correlate to levels of gene expression, and this study utilizes ChIP-seq to classify activating H3K4me3 and silencing H3K27me3 marks on a genome-wide scale for soil-grown mature and naturally senescent Arabidopsis leaves. ChIPnorm was used to normalize data sets and identify genomic regions with significant differences in the two histone methylation patterns, and the differences were correlated to changes in gene expression. Genes that showed an increase in the H3K4me3 mark in older leaves were senescence up-regulated, while genes that showed a decrease in the H3K4me3 mark in the older leaves were senescence down-regulated. For the H3K27me3 modification, genes that lost the H3K27me3 mark in older tissue were senescence up-regulated. Only a small number of genes gained the H3K27me3 mark, and these were senescence down-regulated. Approximately 50% of senescence up-regulated genes lacked the H3K4me3 mark in both mature and senescent leaf tissue. Two of these genes, SAG12 and At1g73220, display strong senescence up-regulation without the activating H3K4me3 histone modification. This study provides an initial epigenetic framework for the developmental transition into senescence.