The photosynthetic CO2-fixation rates, chlorophyll content, chloroplast ultrastructure and other leaf characteristics (e.g. variable fluorescence, stomata density, soluble carbohydrate content) were studied in a comparative way in sun and shade leaves of beech (Fagus sylvatica) and in high-light and low-light seedlings. 1. Sun leaves of the beech possess a smaller leaf area, higher dry weight, lower water content, higher stomata density, higher chlorophyll a/b ratios and are thicker than the shade leaves. Sun leaves on the average contain more chlorophyll in a leaf area unit; the shade leaf exhibits more chlorophyll on a dry weight basis. Sun leaves show higher rates for dark respiration and a higher light saturation of photosynthetic CO2-fixation. Above 2000 lux they are more efficient in photosynthetic quantum conversion than the shade leaves. 2. The development of HL-radish plants proceeds much faster than that of LL-plants. The cotyledons of HL-plants show a higher dry weight, lower water content, a higher ratio of chlorophyll a/b and a higher gross photosynthesis rate than the cotyledons of the LL-plants, which possess a higher chlorophyll content per dry weight basis. The large area of the HL-cotyledon on the one hand, as well as the higher stomata density and the higher respiration rate in the LL-cotyledon on the other hand, are not in agreement with the characteristics of sun and shade leaves respectively. 3. The development, growth and wilting of wheat leaves and the appearance of the following leaves (leaf succession) is much faster at high quanta fluence rates than in weak light. The chlorophyll content is higher in the HL-leaf per unit leaf area and in the LL-leaf per g dry weight. There are no differences in the stomata density and leaf area between the HL- and LL-leaf. There are fewer differences between HL- and LL-leaves than in beech or radish leaves. 4. The chloroplast ultrastructure of shade-type chloroplasts (shade leaves, LL-leaves) is not only characterized by a much higher number of thylakoids per granum and a higher stacking degree of thylakoids, but also by broader grana than in sun-type chloroplasts (sun leaves, HL-leaves). The chloroplasts of sun leaves and of HL-leaves exhibit large starch grains. 5. Shade leaves and LL-leaves exhibit a higher maximum chlorophyll fluorescence and it takes more time for the fluorescence to decline to the steady state than in sun and HL-leaves. The variable fluorescence VF (ratio of fluorescence decrease to steady state fluorescence) is always higher in the sun and HL-leaf of the same physiological stage (maximum chlorophyll content of the leaf) than in the shade and LL-leaf. The fluorescence emission spectra of sun and HL-leaves show a higher proportion of chlorophyli fluorescence in the second emission maximum F2 than shade and LL-leaves. 6. The level of soluble carbohydrates (reducing sugars) is significantly higher in sun and HL-leaves than in shade and LL-leaves and even reflects changes in the amounts of the daily incident light. 7. Some but not all...

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Plants are capable of synthesizing a myriad of isoprenoids and prenyl lipids. Much attention has been focused on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the enzyme that synthesizes mevalonate and is generally considered responsible for the regulation of substrate flux to isoprenoids. In contrast to vertebrates, where there seems to exist only one HMGR gene, in plants a small family of isogenes appears differentially expressed in regard to location and time. Much less is known in plants about the preceding steps, viz. the conversion of acetyl-CoA to HMG-CoA. An enzyme system has been isolated from radish that can catalyze this transformation, and which shows some unusual properties in vitro. The intracellular localization of the early steps of isoprenoid biosynthesis in plant cells is still a matter of debate. The various observations and hypotheses derived from incorporation and inhibition studies are somewhat contradictory, and an attempt is being made to rationalize various findings that do not at first seem compatible. There are good arguments in favor of an exclusively cytoplasmic formation of isopentenyl pyrophosphate (IPP) via mevalonic acid, but other studies and observations suggest an independent formation in plastids. Other possibilities are being considered, such as the existence of independent (compartmentalized) biosynthetic pathways of IPP formation via the so-called Rohmer pathway. Substrate channeling through the formation of end product-specific multienzyme complexes (metabolons) with no release of substrate intermediates will also be discussed.

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To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [14 C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.In higher plants, two distinct pathways have been shown to operate concomitantly for synthesizing isopentenyl diphosphate, the common precursor for all isoprenoids. Plastid isoprenoids such as carotenoids, mono-and diterpenes, or the prenyl chains of chlorophylls and plastoquinones are formed from 2-Cmethyl-d-erythritol 4-phosphate, which itself arises from the initial condensation of pyruvate with glyceraldehyde 3-phosphate (for review, see Lichtenthaler, 1999; Rohmer, 1999). In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate (MVA) pathway, in which 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase (HMGR) plays a key role. This enzyme is encoded by a multigene family (Bach et al., 1991; Stermer et al., 1994). In this pathway, farnesyl diphosphate (FPP) occupies a central position from which specific cis-and trans-prenyltransferases dispatch isoprene units to either sterols or non-sterol isoprenoids as represented by sesquiterpenes, ubiquinone, heme a, polyprenols, or prenylated proteins (Fig. 1). It has been recently proposed that specific classes of isoprenoids might be produced within distinct metabolic channels or metabolons, probably involving individual H...

SummaryBrassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N ⁄ S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N ⁄ S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H 2 O 2 )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes.