Christian freedom according to Paul

This thesis is an exegetical survey of those passages in Paul in which the Apostle demonstrates his understanding of the freedom wrought in Christ, whether or not the idea of freedom is explicit or a member of the ελευٯερία word-group is in evidence. The thesis is divided into three sections, dealing with Paul's understanding of freedom in relation to the law, to sin and to death. The first theme is subdivided into sections dealing with God's condemnation of sin in the law. His act of liberation in Christ, and the consequences of this act for the believer's life and freedom. Evidence is produced to show that the law plays a continuing positive role in the life of the believer, according to Paul. In Christ the believer is freed from the law's just condemnation, but freed for obedience to the law's righteous command. This freedom must be expressed in the form of obedience, not licence. In the second theme, freedom in relation to sin, a number of passages are examined in which the Apostle demonstrates his understanding of the nature and extent of freedom from sin as a result of the work of Christ. The thesis seeks to show the seriousness of the struggle against sin, and the way in which the believer, while receiving a genuine measure of freedom in Christ, fails woefully to live up to God's righteous standard shown in his law. Freedom from sin's dominion is freedom for obedience, and this obedience is to be expressed in the form of slavery to righteousness. In the final theme, that of freedom in relation to death, a number of passages are examined which show the same pattern of genuine but incomplete freedom in this area, and how the freedom from death given in Christ is real, though incomplete in this life, and should result in freedom for a life lived in obedience to God and to His righteous commands

Ascospore release and survival in Sclerotinia sclerotiorum

The release and survival of ascospores of a UK Sclerotinia sclerotiorum isolate were studied. Apothecia placed in a spore clock apparatus with different lighting regimes at 15 °C released ascospores continuously with an increasing rate for the duration of experiments (72–84 h). Spore release was not confined to light or dark periods in alternating regimes and occurred in continuous dark or light. Ascospores were released in both saturated air (90–95% rh) and at 65–75% rh. High temperature and rh were detrimental to ascospore survival but spore viability was maintained for longer periods than previously reported. The significance of these results in relation to disease control is discussed

Investigation of salicylate hepatic responses in comparison with chemical analogues of the drug

AbstractAnti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling

Metformin selectively targets redox control of complex I energy transduction

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD+ couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD+ couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled

A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growth

[EN] Adverse environmental conditions trigger responses in plants that promote stress tolerance and survival at the expense of growth(1). However, little is known of how stress signalling pathways interact with each other and with growth regulatory components to balance growth and stress responses. Here, we show that plant growth is largely regulated by the interplay between the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1) protein kinase and the abscisic acid (ABA) phytohormone pathway. While SnRK2 kinases are main drivers of ABA-triggered stress responses, we uncover an unexpected growth-promoting function of these kinases in the absence of ABA as repressors of SnRK1. Sequestration of SnRK1 by SnRK2-containing complexes inhibits SnRK1 signalling, thereby allowing target of rapamycin (TOR) activity and growth under optimal conditions. On the other hand, these complexes are essential for releasing and activating SnRK1 in response to ABA, leading to the inhibition of TOR and growth under stress. This dual regulation of SnRK1 by SnRK2 kinases couples growth control with environmental factors typical for the terrestrial habitat and is likely to have been critical for the water-to-land transition of plants.We thank J.-K. Zhu for the snrk2 mutants, M. Bennett for the SnRK2.2-GFP line, C. Koncz for the SnRK1-GFP line, X. Li for the SnRK2.3-FLAG OE line, J. Schroeder for the GFP-His-FLAG and SnRK2.6-His-FLAG OE lines, C. Mackintosh for the TPS5 antibody and the Nottingham Arabidopsis stock centre for T-DNA mutant seeds. The IGC Plant Facility (Vera Nunes) is thanked for excellent plant care. This work was supported by Fundacao para a Ciencia e a Tecnologia through the R&D Units UIDB/04551/2020 (GREEN-IT-Bioresources for Sustainability) and UID/MAR/04292/2019, FCT project nos. PTDC/BIA-PLA/7143/2014, LISBOA-01-0145-FEDER-028128 and PTDC/BIA-BID/32347/2017, and FCT fellowships/contract nos. SFRH/BD/122736/2016 (M.A.), SFRH/BPD/109336/2015 (A.C.), PD/BD/150239/2019 (D.R.B.), and IF/00804/2013 (E.B.G.). Work in P.L.R.'s laboratory was funded by MCIU grant no. BIO2017-82503-R. C.M. thanks the LabEx Paris Saclay Plant Sciences-SPS (ANR-10-LABX-040-SPS) for support. B.B.P. was funded by Programa VALi+d GVA APOSTD/2017/039. This project has received funding from the European Union Horizon 2020 research and innovation programme (grant agreement no. 867426-ABA-GrowthBalance-H2020-WF-2018-2020/H2020-WF-01-2018, awarded to B.B.P.). This work is dedicated to the memory of our beloved friend and colleague Americo Rodrigues.Belda-Palazón, B.; Adamo, M.; Valerio, C.; Ferreira, LJ.; Confraria, A.; Reis-Barata, D.; Rodrigues, A.... (2020). A dual function of SnRK2 kinases in the regulation of SnRK1 and plant growth. Nature Plants (Online). 6(11):1345-1353. https://doi.org/10.1038/s41477-020-00778-wS13451353611Huot, B., Yao, J., Montgomery, B. L. & He, S. Y. Growth-defense tradeoffs in plants: a balancing act to optimize fitness. Mol. Plant 7, 1267–1287 (2014).Baena-Gonzalez, E., Rolland, F., Thevelein, J. M. & Sheen, J. A central integrator of transcription networks in plant stress and energy signalling. Nature 448, 938–942 (2007).Baena-Gonzalez, E. & Sheen, J. Convergent energy and stress signaling. Trends Plant Sci. 13, 474–482 (2008).Nukarinen, E. et al. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation. Sci. Rep. 6, 31697 (2016).Rodrigues, A. et al. ABI1 and PP2CA phosphatases are negative regulators of Snf1-related protein kinase1 signaling in Arabidopsis. Plant Cell 25, 3871–3884 (2013).Nakashima, K., Yamaguchi-Shinozaki, K. & Shinozaki, K. The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought, cold, and heat. Front. Plant Sci. 5, 170 (2014).Fujii, H., Verslues, P. E. & Zhu, J. K. Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. Plant Cell 19, 485–494 (2007).Mustilli, A. C., Merlot, S., Vavasseur, A., Fenzi, F. & Giraudat, J. Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14, 3089–3099 (2002).Umezawa, T. et al. Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proc. Natl Acad. Sci. USA 106, 17588–17593 (2009).Vlad, F. et al. Protein phosphatases 2C regulate the activation of the Snf1-related kinase OST1 by abscisic acid in Arabidopsis. Plant Cell 21, 3170–3184 (2009).Yoshida, R. et al. The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J. Biol. Chem. 281, 5310–5318 (2006).Ma, Y. et al. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324, 1064–1068 (2009).Park, S. Y. et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324, 1068–1071 (2009).Bitrian, M., Roodbarkelari, F., Horvath, M. & Koncz, C. BAC-recombineering for studying plant gene regulation: developmental control and cellular localization of SnRK1 kinase subunits. Plant J. 65, 829–842 (2011).Jossier, M. et al. SnRK1 (SNF1-related kinase 1) has a central role in sugar and ABA signalling in Arabidopsis thaliana. Plant J. 59, 316–328 (2009).Lin, C. R. et al. SnRK1A-interacting negative regulators modulate the nutrient starvation signaling sensor SnRK1 in source-sink communication in cereal seedlings under abiotic stress. Plant Cell 26, 808–27 (2014).Lu, C. A. et al. The SnRK1A protein kinase plays a key role in sugar signaling during germination and seedling growth of rice. Plant Cell 19, 2484–2499 (2007).Radchuk, R. et al. Sucrose non-fermenting kinase 1 (SnRK1) coordinates metabolic and hormonal signals during pea cotyledon growth and differentiation. Plant J. 61, 324–338 (2010).Radchuk, R., Radchuk, V., Weschke, W., Borisjuk, L. & Weber, H. Repressing the expression of the SUCROSE NONFERMENTING-1-RELATED PROTEIN KINASE gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype. Plant Physiol. 140, 263–278 (2006).Tsai, A. Y. & Gazzarrini, S. AKIN10 and FUSCA3 interact to control lateral organ development and phase transitions in Arabidopsis. Plant J. 69, 809–821 (2012).Tsai, A. Y. & Gazzarrini, S. Trehalose-6-phosphate and SnRK1 kinases in plant development and signaling: the emerging picture. Front. Plant Sci. 5, 119 (2014).Zhang, Y. et al. Arabidopsis sucrose non-fermenting-1-related protein kinase-1 and calcium-dependent protein kinase phosphorylate conserved target sites in ABA response element binding proteins. Ann. Appl. Biol. 153, 401–409 (2008).Ramon, M. et al. Default activation and nuclear translocation of the plant cellular energy sensor SnRK1 regulate metabolic stress responses and development. Plant Cell 31, 1614–1632 (2019).Lopez-Molina, L., Mongrand, S. & Chua, N. H. A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis. Proc. Natl Acad. Sci. USA 98, 4782–4787 (2001).Garcia, D. & Shaw, R. J. AMPK: mechanisms of cellular energy sensing and restoration of metabolic balance. Mol. Cell 66, 789–800 (2017).Dobrenel, T. et al. The Arabidopsis TOR kinase specifically regulates the expression of nuclear genes coding for plastidic ribosomal proteins and the phosphorylation of the cytosolic ribosomal protein S6. Front. Plant Sci. 7, 1611 (2016).Wang, P. et al. Reciprocal regulation of the TOR kinase and ABA receptor balances plant growth and stress response. Mol. Cell 69, 100–112 e106 (2018).Van Leene, J. et al. Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase. Nat. Plants 5, 316–327 (2019).Dietrich, D. et al. Root hydrotropism is controlled via a cortex-specific growth mechanism. Nat. Plants 3, 17057 (2017).Wu, Q. et al. Ubiquitin ligases RGLG1 and RGLG5 regulate abscisic acid signaling by controlling the turnover of phosphatase PP2CA. Plant Cell 28, 2178–2196 (2016).Belin, C. et al. Identification of features regulating OST1 kinase activity and OST1 function in guard cells. Plant Physiol. 141, 1316–1327 (2006).Fujii, H. & Zhu, J. K. Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc. Natl Acad. Sci. USA 106, 8380–8385 (2009).Fujita, Y. et al. Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis. Plant Cell Physiol. 50, 2123–2132 (2009).Nakashima, K. et al. Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol. 50, 1345–1363 (2009).Fujii, H. et al. In vitro reconstitution of an abscisic acid signalling pathway. Nature 462, 660–664 (2009).Shen, W., Reyes, M. I. & Hanley-Bowdoin, L. Arabidopsis protein kinases GRIK1 and GRIK2 specifically activate SnRK1 by phosphorylating its activation loop. Plant Physiol. 150, 996–1005 (2009).Cheng, C. et al. SCFAtPP2-B11 modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana. PLoS Genet. 13, e1006947 (2017).Harthill, J. E. et al. Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose. Plant J. 47, 211–223 (2006).Song, Y. et al. Identification of novel interactors and potential phosphorylation substrates of GsSnRK1 from wild soybean (Glycine soja). Plant Cell Environ. 42, 145–157 (2018).Wang, X., Du, Y. & Yu, D. Trehalose phosphate synthase 5-dependent trehalose metabolism modulates basal defense responses in Arabidopsis thaliana. J. Integr. Plant Biol. 61, 509–527 (2019).Broeckx, T., Hulsmans, S. & Rolland, F. The plant energy sensor: evolutionary conservation and divergence of SnRK1 structure, regulation, and function. J. Exp. Bot. 67, 6215–6252 (2016).Wang, Y. et al. AKINbeta1, a subunit of SnRK1, regulates organic acid metabolism and acts as a global modulator of genes involved in carbon, lipid, and nitrogen metabolism. J. Exp. Bot. 71, 1010–1028 (2020).Yoshida, T. et al. The role of abscisic acid signaling in maintaining the metabolic balance required for Arabidopsis growth under nonstress conditions. Plant Cell 31, 84–105 (2019).Zheng, Z. et al. The protein kinase SnRK2.6 mediates the regulation of sucrose metabolism and plant growth in Arabidopsis. Plant Physiol. 153, 99–113 (2010).Cutler, S. R., Rodriguez, P. L., Finkelstein, R. R. & Abrams, S. R. Abscisic acid: emergence of a core signaling network. Annu Rev. Plant Biol. 61, 651–679 (2010).Kravchenko, A. et al. Mutations in the Arabidopsis Lst8 and Raptor genes encoding partners of the TOR complex, or inhibition of TOR activity decrease abscisic acid (ABA) synthesis. Biochem. Biophys. Res. Commun. 467, 992–997 (2015).Salem, M. A., Li, Y., Wiszniewski, A. & Giavalisco, P. Regulatory-associated protein of TOR (RAPTOR) alters the hormonal and metabolic composition of Arabidopsis seeds, controlling seed morphology, viability and germination potential. Plant J. 92, 525–545 (2017).Bakshi, A. et al. Ectopic expression of Arabidopsis target of rapamycin (AtTOR) improves water-use efficiency and yield potential in rice. Sci. Rep. 7, 42835 (2017).De Smet, I. et al. An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis. Plant J. 33, 543–555 (2003).Hrabak, E. M. et al. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132, 666–680 (2003).Hauser, F., Waadt, R. & Schroeder, J. I. Evolution of abscisic acid synthesis and signaling mechanisms. Curr. Biol. 21, R346–R355 (2011).Umezawa, T. et al. Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport. Plant Cell Physiol. 51, 1821–1839 (2010)