Extracellular invertase is an essential component of cytokinin-mediated delay of senescence

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.     

Lipase-catalyzed synthesis of chlorogenate fatty esters in solvent-free medium

Chlorogenic acid (5-caffeoylquinic acid or 5-CQA) is an hydrophilic phenolic compound with antioxidant properties. Because of its high polarity, its antioxidant properties may be altered when formulated in oil based food or cosmetic preparations. Therefore, there is an interest in trying to enhance its hydrophobicity by grafting of an aliphatic chain. Such lipophilization reactions can be generally achieved through enzymatic catalysis. Our study consisted in synthesizing fatty cholorogenate esters in a two steps reaction. Firstly, 5-CQA was chemically esterified by methanol using an Amberlite IR120 H resin to obtain methyl chlorogenate that is more soluble in the fatty alcohols than 5-CQA. Secondly, this chlorogenate intermediate was transesterified with fatty alcohols of various chain lengths (C₄, C₈, C₁₂, or C₁₆) in the presence of Candida antarctica B lipase. Under optimal reaction conditions (a_w = 0.05; 5% (w/w) of biocatalyst), the transesterification rates were until two-fold higher than in the direct lipase-catalyzed esterification of chlorogenic acid by the same alcohols. The two-step reaction overall yield was between 61 and 93% depending on the alcohol chain length, whereas it was 40–60% for the direct esterification with the same alcohols.     

Toward the Synthesis of Pyroglutamate Lauroyl Ester: Biocatalysis Versus Chemical Catalysis

The synthesis of dodecyl pyroglutamate (or pyroglutamate lauroyl ester) was achieved in a two-step process involving a pyroglutamic acid alkyl ester intermediate. The reaction was carried out either by lipase or by chemical catalysis using ion exchange resin. Among the various tested lipases, the one from Candida antarctica B gave the best results allowing 73% formation of the desired ester after 6 h. Comparing the efficiency of this latter lipase with the one of Amberlyst IR120H resin in catalyzing this reaction, the biocatalyst gave a molar yield of pyroglutamate lauroyl ester of 79% compared to 69% when using the ion exchange resin starting with 1.04 mmol substrate in each case.     

Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.     

6-Amino-2-mercapto-3H-pyrimidin-4-one derivatives as new candidates for the antagonism at the P2Y12 receptors

P2Y₁₂ plays an important role in platelet aggregation, which makes it an interesting target for antithrombotic agents. Compounds that antagonize P2Y₁₂ include the active metabolites of thienopyridines and molecules that are structurally related to ATP, which is an antagonist of P2Y₁₂. During the last few years, our group has been working on the development of P2Y₁₂ receptors antagonists that are based on an extremely simple chemical structure, the 6-amino-2-mercapto-3H-pyrimidin-4-one, variously substituted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two known P2Y₁₂ antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light transmission aggregometry. None of the tested compounds induced platelet aggregation, while some of them, at concentration of 10^?4 M, partially inhibited platelet aggregation induced by ADP 10^?6 M. The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the forskolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y₁₂-receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of P2 receptors, and in particular of P2Y₁₂ and suggest that further development of this structurally new series of compounds as P2Y₁₂ receptors antagonists is recommended.