Chalcone synthase [naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), E.C. 2.3.1.74], the key enzyme of flavonoid pathways that was believed to be soluble, has been localized on ribosome-bearing endoplasmic reticulum membranes in the epidermis of buckwheat (Fagopyrum esculentum M.) hypocotyls. Enzyme activity measurement and immunoblots of buckwheat hypocotyl homogenates that were fractionated on linear sucrose density gradients and developed with a specific chalcone synthase antibody and a 20-nm ImmunoGold coijugate showed the presence of chalcone synthase in fractions enriched in endoplasmic reticulum membranes. The presence of chalcone synthase on these membranes was not caused by nonspecific adsorption or entrapment of proteins. Immunocytochemical investigations with both a 5-nm and a 20-nm ImmunoGold conjugate showed that chalcone synthase was associated with the cytoplasmic face of rough (ribosome bearing) endoplasmic reticulum membranes. Plasma membrane, nucleus, plastids, mitochondria, golgi, and the tonoplast were not labeled. These data are consistent with our earlier described model suggesting that the synthesis of phenylpropanoids and flavonoids takes place partially or fully on membrane-associated enzyme complexes.Flavonoid compounds are produced by most, if not all, higher plants and are synthesized from p-coumaryl-CoA and malonyl-CoA by the enzyme chalcone synthase [CHS; naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), EC 2.3.1.74]. Cell fractionation experiments showed that the majority of the activity of this enzyme was "soluble," with small but significant portions associated with a membrane fraction (1). Ultracentrifugation (2) and gel-filtration experiments (3) suggested that CHS is associated with the endoplasmic reticulum (ER) membranes. Convincing evidence exists that another soluble enzyme of this pathway, phenylalanine ammonia lyase (E.C. 4.3.1.5) is at least in part membrane associated (4).Detection of enzyme activities in the soluble cytoplasmic fraction after cell fractionation is not a convincing argument for subcellular localization in vivo. Most homogenization procedures destroy cellular organization, and most suspension media that are used during cell fractionations are not physiological in nature. It is becoming increasingly clear that many so-called cytosolic enzymes interact with each other and the structural components of the cell, and the enzyme activities are modified by these interactions (5). Thus, the term "soluble enzyme" should not be taken as indicating that the catalytically active compartment is a true solution. Rather, it should be regarded as an operational definition, referring more to the result of homogenization and centrifugation procedures in subcellular fractionation than to any established in vivo characteristic.Our previous studies on localizing weakly associated enzymes or ER membranes by activity alone were greatly hampered by inactivation of enzymes during the isolati...

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Vaccinium myrtillus L. is a common shrub in the herbaceous layer of pine forests. In connection with resistance to pollution it has been widely used in the environmental monitoring based on heavy metal accumulations in the foliage. The present study investigates leaves of bilberry growing under influence of emissions from a Zn-Pb smelter. Determination of heavy metal levels by AAS revealed enhanced accumulations of Zn, Pb, Cd, and Fe, and the level of Zn fell into the toxic range compiled for plants of average sensitivity. To verify the significance of metal accumulations occurring in the studied plant, the concentrations of phenolics were quantified by measuring UV absorption of foliage extracts. Phenolics can be used as a potential biomarker of pollution because they participate in plant's response to accumulation of heavy metals, acting as antioxidants able to scavenge free radicals produced by metal ions. Leaves collected near the smelter contained significantly higher amounts of phenolics. Since these compounds are also responsible for ecological interactions, changes in their concentrations resulting from heavy metal accumulations might also influence other organisms in the ecosystem. To study the role of phenolics in heavy metal accumulations at the cellular level, a special fixation was applied for TEM, involving glutaraldehyde with caffeine, to precipitate phenolics in the cell. A histochemical reaction showed significantly higher electron-opacity, most probably deposits of phenolics, in cells of the leaves collected near the smelter. The subsequent degenerative changes in mesophyll of these leaves were characterized by increased contents of heterogeneous multivesicular structures, degeneration of chloroplasts, and disorganisation of membranes leading to death of the cell and necrosis.

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Achenes of Tragopogon dubius have previously been described as dimorphic with respect to size and colour. In this investigation we found that there is a continuum of weight change from lighter central achenes to the heavier peripheral achenes. The main component of change is the pericarp. Outer achenes may also contain larger quantities of phenolic compounds than inner achenes. The differences in mass, morphology, and chemical characteristics may be significant enough to result in differences in germination behaviour. No such differences were seen in standard laboratory germination tests, though in a greenhouse experiment, seedlings from peripheral achenes emerged later and were larger and taller than seedlings from central achenes. The presence of phenolic compounds in the fruits of T. dubius suggests that the peripheral morphs may retain viability longer in the seed bank. Key words: somatic polymorphism, heteromorphism, germination, emergence, Tragopogon.