Trichomes of tobacco excrete zinc as zinc-substituted calcium carbonate and other zinc-containing compounds

Tobacco (Nicotiana tabacum L. cv Xanthi) plants were exposed to toxic levels of zinc (Zn). Zn exposure resulted in toxicity signs in plants, and these damages were partly reduced by a calcium (Ca) supplement. Confocal imaging of intracellular Zn using Zinquin showed that Zn was preferentially accumulated in trichomes. Exposure to Zn and Zn + Ca increased the trichome density and induced the production of Ca/Zn mineral grains on the head cells of trichomes. These grains were aggregates of submicrometer-sized crystals and poorly crystalline material and contained Ca as major element, along with subordinate amounts of Zn, manganese, potassium, chlorine, phosphorus, silicon, and magnesium. Micro x-ray diffraction revealed that the large majority of the grains were composed essentially of metal-substituted calcite (CaCO3). CaCO3 polymorphs (aragonite and vaterite) and CaC2O4 (Ca oxalate) mono- and dihydrate also were identified, either as an admixture to calcite or in separate grains. Some grains did not diffract, although they contained Ca, suggesting the presence of amorphous form of Ca. The presence of Zn-substituted calcite was confirmed by Zn K-edge micro-extended x-ray absorption fine structure spectroscopy. Zn bound to organic compounds and Zn-containing silica and phosphate were also identified by this technique. The proportion of Zn-substituted calcite relative to the other species increased with Ca exposure. The production of Zn-containing biogenic calcite and other Zn compounds through the trichomes is a novel mechanism involved in Zn detoxification. This study illustrates the potential of laterally resolved x-ray synchrotron radiation techniques to study biomineralization and metal homeostasis processes in plants.     

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters. Part II. (Oxy)-sitosterol esters derived from oleic acid and from 9,10-dihydroxystearic acid [1

Several efficient synthetic routes giving readily access to (oxy)-sitosterol esters and (oxy)-cholesterol esters derived respectively from oleic acid and from 9,10-dihydroxystearic acid were developed for the first time. This approach allowed that sufficient amounts of the latter were available in order to carry out further biological studies.     

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.     

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters Part I. Regioselective hydrogenation of stigmasterol: an easy access to oxyphytosterols

The synthesis of several oxyphytosterols is described starting from stigmasterol, the key step being the regioselective hydrogenation of the 22-23 double bond of the latter.     

Repression of O-methyltransferase genes in transgenic tobacco affects lignin synthesis and plant growth.

Among the different enzymatic steps leading to lignin biosynthesis, two methylation reactions introduce the methyl groups borne by guaiacyl (G) and syringyl (S) units. Tobacco possesses a complex system of methylation comprising three classes of CCoAOMTs (caffeoyl-CoA-O-methyltransferases) and two classes of COMTs (caffeic acid OMTs). Antisense plants transformed with the CCoAOMT sequence alone or fused to COMT I sequence have been produced and compared to ASCOMT I plants in order to study the specific role of each OMT isoform in lignin biosynthesis, plant development and resistance to pathogens. Tobacco plants strongly inhibited in OMT activities have been selected and analyzed. Whereas antisense COMT I plants exhibited no visual phenotype, CCoAOMT repression was shown to strongly affect the development of both single and double transformants: a reduction of plant growth and the alteration of flower development were observed in the most inhibited plants. Lignin analysis performed by Klason and thioacidolysis methods, showed a decrease in the lignin quantity and changes in the lignin structure of ASCCoAOMT and ASCCoAOMT/ASCOMT I transgenics but not in ASCOMT I plants. Inhibition of COMT I in single as well as in double transformed tobacco was demonstrated to decrease S unit synthesis and to provoke the accumulation of 5-hydroxyguaiacyl lignin units. ASCCoAOMT/ASCOMT I tobacco was affected in lignin amount and composition, thus demonstrating additive effects of inhibition of both enzymes. The changes of lignin profiles and the phenotypical and molecular alterations observed in the different transgenic lines were particularly prominent at the later stages of plant development.     

Use of Green Fluorescent Protein To Tag Lactic Acid Bacterium Strains under Development as Live Vaccine Vectors

The lactic acid bacteria (LAB) are safe microorganisms which are mainly used for the preparation of fermented foods and for probiotic applications. The potential of LAB as live vehicles for the production and delivery of therapeutic molecules such as antigens is also being actively investigated today. However, very little is known about the fate of live LAB when administered in vivo and about the interaction of these microorganisms with the nasal or gastrointestinal ecosystem. For future applications, it is essential to be able to discriminate the biotherapeutic strain from the endogenous microflora and to unravel the mechanisms underlying the postulated health-beneficial effect. We therefore started to investigate both aspects in a mouse model with two LAB species presently under development as live vaccine vectors, i.e., Lactococcus lactis and Lactobacillus plantarum. We have constructed different expression vectors carrying the gfp (green fluorescent protein [GFP]) gene from the jellyfish Aequoria victoria, and we found that this visible marker was best expressed when placed under the control of the inducible strong nisA promoter from L. lactis. Notably, a threshold amount of GFP was necessary to obtain a bright fluorescent phenotype. We further demonstrated that fluorescent L. plantarum NCIMB8826 can be enumerated and sorted by flow cytometry. Moreover, tagging of this strain with GFP allowed us to visualize its phagocytosis by macrophages in vitro and ex vivo and to trace it in the gastrointestinal tract of mice upon oral administration.     

Purification and properties of the three o-diphenol-O-methyltransferases of tobacco leaves

Three o-diphenol-O-methyltransferases (OMTs I, II and III) which catalyse the monomethylation of various o-diphenols using S-adenosyl-L-methionine as methyl donor were isolated and purified about 210-, 70-, and 70-fold, respectively, from leaves of Nicotiana tabacum cv Samsun NN. They had slightly different MWs (93 000, 90 000 and 100000 for OMTs 1, 11 and Ill respectively) and slightly different pls (5.21, 4.80 and 4.74). The activities of all three enzymes were very stable when stored at 0° but they had different sensitivities to ultrafiltration and to heat treatment (45°). In none of the enzymes was there any change in reaction rate when Mg²⁺ ions or EDTA were added. The three enzymes exhibited very high and similar affinities towards the substrate S-adenosylmethionine and the reaction product S-adenosylhomocysteine, but they differed markedly in specificities towards the various o-diphenolic substrates. Relative methylation efficiencies were estimated from the calculation of the V/K_m ratios that led to the following decreasing order of best substrates: 5-hydroxyferulic acid > caffeic acid > homo-catechol > esculetin > protocatechuic aldehyde > catechol > hydrocaffeic acid > chlorogenic acid, for OMT I, and: homocatechol > catechol > protocatechuic aldehyde > esculetin ≈ cafreic acid > 5-hydroxyferulic acid, for both OMTsIIandIII. Most of the o-diphenols assayed were methylated exclusively in the meta position, but all three tobacco OMTs showed both para and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and escultin. Since K_m values towards the two position of methylation were always found to be identical, we conclude that each enzyme bears only one catalytic site.