Thrifty metabolism that favors fat storage after caloric restriction: a role for skeletal muscle phosphatidylinositol-3-kinase activity and AMP-activated protein kinase.

Energy conservation directed at accelerating body fat recovery (or catch-up fat) contributes to obesity relapse after slimming and to excess fat gain during catch-up growth after malnutrition. To investigate the mechanisms underlying such thrifty metabolism for catch-up fat, we tested whether during refeeding after caloric restriction rats exhibiting catch-up fat driven by suppressed thermogenesis have diminished skeletal muscle phosphatidylinositol-3-kinase (PI3K) activity or AMP-activated protein kinase (AMPK) signaling-two pathways required for hormone-induced thermogenesis in ex vivo muscle preparations. The results show that during isocaloric refeeding with a low-fat diet, at time points when body fat, circulating free fatty acids, and intramyocellular lipids in refed animals do not exceed those of controls, muscle insulin receptor substrate 1-associated PI3K activity (basal and in vivo insulin-stimulated) is lower than that in controls. Isocaloric refeeding with a high-fat diet, which exacerbates the suppression of thermogenesis, results in further reductions in muscle PI3K activity and in impaired AMPK phosphorylation (basal and in vivo leptin-stimulated). It is proposed that reduced skeletal muscle PI3K/AMPK signaling and suppressed thermogenesis are interdependent. Defective PI3K or AMPK signaling will reduce the rate of substrate cycling between de novo lipogenesis and lipid oxidation, leading to suppressed thermogenesis, which accelerates body fat recovery and furthermore sensitizes skeletal muscle to dietary fat-induced impairments in PI3K/AMPK signaling.

Isolation and characterization of signal transduction mutants of Arabidopsis thaliana that constitutively activate the octadecanoid pathway and form necrotic microlesions.

Thionins are a group of antimicrobial polypeptides that form part of the plant's defense mechanism against pathogens. The Thi 2.1 thionin gene of Arabidopsis thaliana has been shown to be inducible by jasmonic acid (JA), an oxylipin-like hormone derived from oxygenated linolenic acid and synthesized via the octadecanoid pathway. The JA-dependent regulation of the Thi 2.1 gene has been exploited for setting up a genetic screen for the isolation of signal transduction mutants that constitutively express the Thi 2.1 gene. Ten cet-mutants have been isolated which showed a constitutive expression of the thionin gene. Allelism tests revealed that they represent at least five different loci. Some mutants are dominant, others recessive, but all cet mutations behaved as monogenic traits when backcrossed with Thi 2.1-GUS plants. Some of the mutants overproduce JA and its bioactive precursor 12-oxophytodienoic acid (OPDA) up to 40-fold while others have the same low levels as the control wildtype plants. Two of the mutants showed a strong induction of both the salicylic acid (SA)- and the JA-dependent signaling pathways, while the majority seems to be affected only in the octadecanoid pathway. The Thi 2.1 thionin gene and the Pdf 1.2 defensin gene are activated independently, though both are regulated by JA. The cet-mutants, except for one, also show a spontaneous leaf cell necrosis, a reaction often associated with the systemic acquired resistance (SAR) pathway.

Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions.

A major structural component of the cuticle of plants is cutin. Analysis of the function of cutin in vivo has been limited because no mutants with specific defects in cutin have been characterized. Therefore, transgenic Arabidopsis plants were generated that express and secrete a cutinase from Fusarium solani f sp pisi. Arabidopsis plants expressing the cutinase in the extracellular space showed an altered ultrastructure of the cuticle and an enhanced permeability of the cuticle to solutes. In addition, pollen could germinate on fully differentiated leaves of cutinase-expressing plants but not on control leaves. These differences coincided with strong postgenital organ fusions. The junctions of the fusions contained pectic polysaccharides. As fused organs grew apart from each other, organ deformations and protrusions of epidermal cells developed at positions with high mechanical stress. These results demonstrate that an intact cutin layer not only is important for plant-environment interactions but also prevents fusions between different plant organs and is therefore necessary for normal epidermal differentiation and organ formation.

Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway in bean.

Root colonization by specific nonpathogenic bacteria can induce a systemic resistance in plants to pathogen infections. In bean, this kind of systemic resistance can be induced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 and depends on the production of salicylic acid by this strain. In a model with plants grown in perlite we demonstrated that Pseudomonas aeruginosa 7NSK2-induced resistance is equivalent to the inclusion of 1 nM salicylic acid in the nutrient solution and used the latter treatment to analyze the molecular basis of this phenomenon. Hydroponic feeding of 1 nM salicylic acid solutions induced phenylalanine ammonia-lyase activity in roots and increased free salicylic acid levels in leaves. Because pathogen-induced systemic acquired resistance involves similar changes it was concluded that 7NSK2-induced resistance is mediated by the systemic acquired resistance pathway. This conclusion was validated by analysis of phenylalanine ammonia-lyase activity in roots and of salicylic acid levels in leaves of soil-grown plants treated with Pseudomonas aeruginosa. The induction of systemic acquired resistance by nanogram amounts of salicylic acid is discussed with respect to long-distance signaling in systemic acquired resistance.

Glycerol is a suberin monomer. New experimental evidence for an old hypothesis

The monomer composition of the esterified part of suberin can be determined using gas chromatography-mass spectroscopy technology and is accordingly believed to be well known. However, evidence was presented recently indicating that the suberin of green cotton (Gossypium hirsutum cv Green Lint) fibers contains substantial amounts of esterified glycerol. This observation is confirmed in the present report by a sodium dodecyl sulfate extraction of membrane lipids and by a developmental study, demonstrating the correlated accumulation of glycerol and established suberin monomers. Corresponding amounts of glycerol also occur in the suberin of the periderm of cotton stems and potato (Solanum tuberosum) tubers. A periderm preparation of wound-healing potato tuber storage parenchyma was further purified by different treatments. As the purification proceeded, the concentration of glycerol increased at about the same rate as that of alpha,omega-alkanedioic acids, the most diagnostic suberin monomers. Therefore, it is proposed that glycerol is a monomer of suberins in general and can cross-link aliphatic and aromatic suberin domains, corresponding to the electron-translucent and electron-opaque suberin lamellae, respectively. This proposal is consistent with the reported dimensions of the electron-translucent suberin lamellae.

Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis.

Activation of the plant defensin gene PDF1.2 in Arabidopsis by pathogens has been shown previously to be blocked in the ethylene response mutant ein2-1 and the jasmonate response mutant coi1-1. In this work, we have further investigated the interactions between the ethylene and jasmonate signal pathways for the induction of this defense response. Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola led to a marked increase in production of jasmonic acid, and this response was not blocked in the ein2-1 mutant. Likewise, A. brassicicola infection caused stimulated emission of ethylene both in wild-type plants and in coi1-1 mutants. However, treatment of either ein2-1 or coi1-1 mutants with methyl jasmonate or ethylene did not induce PDF1.2, as it did in wild-type plants. We conclude from these experiments that both the ethylene and jasmonate signaling pathways need to be triggered concomitantly, and not sequentially, to activate PDF1.2 upon pathogen infection. In support of this idea, we observed a marked synergy between ethylene and methyl jasmonate for the induction of PDF1.2 in plants grown under sterile conditions. In contrast to the clear interdependence of the ethylene and jasmonate pathways for pathogen-induced activation of PDF1.2, functional ethylene and jasmonate signaling pathways are not required for growth responses induced by jasmonate and ethylene, respectively.

Cutin monomers and surface wax constituents elicit H2O2 in conditioned cucumber hypocotyl segments and enhance the activity of other H2O2 elicitors

Hypocotyls from etiolated cucumber (Cucumis sativus L.) seedlings were gently abraded at their epidermal surface and cut segments were conditioned to develop competence for H2O2 elicitation. Alkaline hydrolysates of cutin from cucumber, tomato, and apple elicited H2O2 in such conditioned segments. The most active constituent of cucumber cutin was identified as dodecan-1-ol, a novel cutin monomer capable of forming hydrophobic terminal chains. Additionally, the cutin hydrolysates enhanced the activity of a fungal H2O2 elicitor, similar to cucumber surface wax, which contained newly identified alkan-1,3-diols. The specificity of elicitor and enhancement activity was further elaborated using some pure model compounds. Certain saturated hydroxy fatty acids were potent H2O2 elicitors as well as enhancers. Some unsaturated epoxy and hydroxy fatty acids were also excellent H2O2 elicitors but inhibited the fungal elicitor activity. Short-chain alkanols exhibited good elicitor and enhancer activity, whereas longer-chain alkan-1-ols were barely active. The enhancement effect was also observed for H2O2 elicitation by ergosterol and chitosan. The physiological significance of these observations might be that once the cuticle is degraded by fungal cutinase, the cutin monomers may act as H2O2 elicitors. Corrosion of cutin may also bring surface wax constituents in contact with protoplasts and enhance elicitation.

The biosynthesis of salicylic acid in potato plants

Spraying potato (Solanum tuberosum L.) leaves with arachidonic acid (AA) at 1500 &mgr;g mL-1 led to a rapid local synthesis of salicylic acid (SA) and accumulation of a SA conjugate, which was shown to be 2-O-beta-glucopyranosylsalicylic acid. Radiolabeling studies with untreated leaves showed that SA was synthesized from phenylalanine and that both cinnamic and benzoic acid were intermediates in the biosynthesis pathway. Using radiolabeled phenylalanine as a precursor, the specific activity of SA was found to be lower when leaves were treated with AA than in control leaves. Similar results were obtained when leaves were fed with the labeled putative intermediates cinnamic acid and benzoic acid. Application of 2-aminoindan-2-phosphonic acid at 40 &mgr;M, an inhibitor of phenylalanine ammonia-lyase, prior to treatment with AA inhibited the local accumulation of SA. When the putative intermediates were applied to leaves in the presence of 2-aminoindan-2-phosphonic acid, about 40% of the expected accumulation of free SA was recovered, but the amount of the conjugate remained constant.

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels.

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.

Gene-Expression Patterns and Levels of Jasmonic Acid in Rice Treated with the Resistance Inducer 2,6-Dichloroisonicotinic Acid.

Acquired disease resistance can be induced in rice (Oryza sativa) by a number of synthetic or natural compounds, but the molecular mechanisms behind the phenomenon are poorly understood. One of the synthetic inducers of resistance, 2,6-dichloroisonicotinic acid (INA), efficiently protected rice leaves from infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. A comparison of gene-expression patterns in plants treated with INA versus plants inoculated with the compatible pathogen M. grisea or the incompatible pathogen Pseudomonas syringae pv syringae revealed only a marginal overlap: 6 gene products, including pathogenesis-related proteins (PR1-PR9), accumulated in both INA-treated and pathogen-attacked leaves, whereas 26 other gene products accumulated only in INA-treated or only in pathogen-attacked leaves. Lipoxygenase enzyme activity and levels of nonconjugated jasmonic acid (JA) were enhanced in leaves of plants treated with a high dose of INA (100 ppm). Exogenously applied JA enhanced the gene induction and plant protection caused by lower doses of INA (0.1 to 10 ppm) that by themselves did not give rise to enhanced levels of endogenous (-)-JA. These data suggest that INA, aside from activating a pathogen-induced signaling pathway, also induces events that are not related to pathogenesis. JA acts as an enhancer of both types of INA-induced reactions in rice.

Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.

Transport of Salicylic Acid in Tobacco Necrosis Virus-Infected Cucumber Plants.

The transport of salicylic acid (SA) was studied in cucumber (Cucumis sativus L.) using 14C-labeled benzoic acid that was injected in the cotyledons at the time of inoculation. Primary inoculation with tobacco necrosis virus (TNV) on the cotyledons led to an induction of systemic resistance of the first primary leaf above the cotyledon against Colletotrichum lagenarium as early as 3 d after inoculation. [14C]SA was detected in the phloem or in the first leaf 2 d after TNV inoculation, whereas [14C]benzoic acid was not detected in the phloem during the first 3 d after TNV inoculation of the cotyledons, indicating phloem transport of [14C]SA from cotyledon. In leaf 1, the specific activity of [14C]SA decreased between 1.7 and 8.6 times compared with the cotyledons, indicating that, in addition to transport, leaf 1 also produced more SA. The amount of SA transported after TNV infection of the cotyledon was 9 to 160 times higher than in uninfected control plants. Thus, SA can be transported to leaf 1 before the development of systemic acquired resistance, and SA accumulation in leaf 1 results both from transport from the cotyledon and from synthesis in leaf 1.

Changing the Dimensions of Suberin Lamellae of Green Cotton Fibers with a Specific Inhibitor of the Endoplasmic Reticulum-Associated Fatty Acid Elongases.

The fibers of the green lint mutant of cotton (Gossypium hirsutum L.) contain large amounts of wax and are suberized. More than 96% of the bifunctional aliphatic suberin monomers ([alpha],[omega]-alkanedioic acids and [omega]-hydroxyalkanoic acids) have chain lengths of C22 and C24 in green cotton fiber suberin. In fibers grown in the presence of S-ethyl-N,N-dipropylthiocarbamate (EPTC), a specific inhibitor of the endoplasmic reticulum-associated fatty acid elongases, the aliphatic suberin monomers were shortened to chain lengths of C16 and C18. Whereas the amounts of most suberin monomers were not negatively affected by the inhibitor treatment, the amounts of [alpha],[omega]-alkanedioic acids and of glycerol were reduced by more than 80%. Analysis in the transmission electron microscope showed a reduction in suberin content after EPTC treatment. The suberin layers were discontinuous and consisted of fewer lamellae than in the controls. A small proportion (up to 22%) of the electron-translucent suberin lamellae were thinner after EPTC treatment, probably because of the shortening of the aliphatic suberin monomers. A larger proportion of the electron-translucent lamellae were thicker than the lamellae in the controls. Possible explanations for this observation are discussed.

Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants.

Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance.

Polysaccharides in the culture medium of cotton (Gossypium hirsutum L.) ovules cultured in vitro.

Polysaccharides secreted into the culture medium (PCM) from cotton (Gossypium hirsutum L.) ovules, culturedin vitro, have been examined. The amount of the polysaccharides increases with the duration of the culture, but except for the galactose content, does not vary markedly with the culture age. Analysis shows that the polysaccharide mixture contains mainly pectins, but there is also a significant amount of xyloglucan. The source of the polysaccharides is most likely the callus tissue which develops on the ovules.

ß-Glucanases in developing cotton (Gossypium hirsutum L.) fibres.

Cotton fibres possess several ß-glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(1→3)-ß-D-glucanase (EC 3.2.1.58) but which also had the characteristics of a ß-glucosidase (EC 3.2.1.21). Endo-(1→3)-ß-D-glucanase activity (EC 3.2.1.39) and much lower levels of (1→4)-ß-D-glucanase activity were also detected. The exo-(1→3)-ß-glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(1→3)-ß-glucanase activity remained constant throughout fibre development. The ß-glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (1→6)-ß-glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O-ß-gentiobiosylglucose.

Changes in the non-structural carbohydrate content of cotton (Gossypium spp.) fibres at different stages of development.

The neutral sugars (glucose, fructose, and sucrose) and the sugar phosphates (glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate) soluble in hot aqueous 80% methanol from the fibres of cotton - Gossypium arboreum L., G. barbadense L., and G. hirsutum L. - were determined at various stages of fibre development. In addition, the (1→3)-ß-D-glucan content was measured and in the case of G. arboreum the rate of (1→3)-ß-D-glucan and cellulose synthesis was determined with [(14)C]sucrose as the precursor. For each of the species a similar chronology was obtained for the changes in content of the various non-structural carbohydrates. At the early stages of secondary wall formation, glucose and fructose exhibited a maximum which was closely followed by a maximum in the (1→3)-ß-D-glucan content and in the sugar phosphates. On the other hand, the sucrose content increased regularly until fibre maturity. The rates of synthesis of (1→3)-ß-D-glucan and of cellulose were highest following the maximum in the (1→3)-ß-D-glucan content, when the latter was being depleted.

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation.

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 µM were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo-ß-1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in ß-1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation ß-1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.

Studies on an immunologic method for the diagnosis of varicose veins.

A glycoprotein fraction was isolated from varicose veins by a successive extraction procedure. Antibodies to this fraction were shown to exist in the serum of persons suffering from varicose veins. The pathology of this illness is discussed in relation to the autoimmune reaction, and the basis for a method for the early diagnosis of varicose veins is proposed.

ß-Glucanases and non-cellulosic glucans in the developing oat plant.

The changes in the levels of various ß-glucan hydrolase activities in the second internode of the stem of the developing oat plant have been examined. Concurrent changes in the non-cellulosic ß-glucans contained in the corresponding total hemicelluloses were also studied. Possible relationships between the observed changes and the growth and development of the plant tissue are discussed.