Thomas J. Bach

Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves

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 characteristics of mature sun and shade leaves are found in HL- and LL-leaves of seedlings. Leaf thickness, dry weight, chlorophyll content, soluble carbohydrate level, photosynthetic CO2-fixation, height and width of grana stacks and starch content, are good parameters to describe the differences between LL- and HL-leaves; with some reservations concerning age and physiological stage of leaf, a/b ratios, chlorophyll content per leaf area unit and the variable fluorescence are also suitable.

Some properties of enzymes involved in the biosynthesis and metabolism of 3-hydroxy-3-methylglutaryl-CoA in plants.

There is hardly a single process in the living plant cell that does not require the biochemical and physiological involvement of isoprenoids. It is thus of great theoretical as well as practical interest to have a profound knowledge of how those various classes of compounds are synthesized, what the properties of the enzymes participating are and how this synthesis is regulated by various factors depending on the demand of the developing and mature plant for various classes of isoprenoids and prenyllipids. Although a large body of information is available on the structures of the myriad of isoprenoids occurring in plants isolated so far, many of the details to be discussed in this contribution have frequently been learned through experiments first performed with animal cells or yeast, systems that, at least as far as the variety of isoprenoid end products is concerned, are not as complex as plants. Therefore, an important purpose of this article is to consider the feasibility of regulatory and of other models developed for non-plant systems that might be likely to explain some features of the enzymology of isoprenoid biosynthesis.

Expression of catalytically active radish 3-hydroxy-3-methylglutaryl coenzyme A reductase in Escherichia coli

Two fragments of a cDNA encoding radish 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET‐8c and expressed in Escherichia coli. The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity. In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form. The amount of soluble active enzyme in cell‐free extracts of E. coli dramatically increased when the temperature during the induction was lowered from 37°C to 22°C.

Mevinolin: A Highly Specific Inhibitor of Microsomal 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase of Radish Plants

The fungal metabolite mevinolin, known to be a potent hypocholesterolemic agent, excerts in vitro a strong inhibitory effect on microsomal HMG-CoA reductase from etiolated radish seedlings at a concentration of about three magnitudes lower than the Km towards the natural substrate (S)-HMG-CoA (I50 = 2.5 × 10-9 ᴍ). Beside this, mevinolin significantly inhibits the root elongation of radish as well as of wheat seedlings already at low concentrations of 10 to 100 ppb (= 2.5 × 10-8 to 2.5 × 10-7 ᴍ).

Brassinosteroids: potent inhibitors of growth of transformed tobacco callus cultures

Abstract The effects of two synthetic brassinosteroids on callus and suspension cultures of Agrobacterium tumefaciens transformed hormone-autonomous Nicotiana tabacum (strain LA6) were examined. Both compounds were potent growth inhibitors at concentrations as low as 10−10 M. Fresh and dry weights and the relative water content of brassinosteroid-treated calli were drastically reduced. The content of soluble proteins and reducing sugars was not affected. The impairment of growth was partially reversible by subculturing calli on BR-free medium but only for material which had been treated with concentrations of BR below 10−8 M. Growth of wild-type, non-transformed tobacco callus cultures (strain SR1) treated with brassinosteroid at different levels of exogenous cytokinin was also severely inhibited. At an intermediate level of BR and low kinetin, however, calli began to differentiate. The simultaneous application of high levels of auxin which otherwise were inhibitory to the transformed (LA6) cells antagnoized, to some extent, the inhibition elicited by BR, in some cases even a stimulation of growth could be observed.

Application of modified Lineweaver-Burk plots to studies of kinetics and regulation of radish 3-hydroxy-3-methylglutaryl-CoA reductase

We propose the use of modified Lineweaver-Burk plots for the correct evaluation of Michaelis-Menten parameters in radioactive enzyme assays. A correction factor X for the translation of 1/S0 into 1/S is directly derived from the integrated Michaelis-Menten equation without the need of complicated calculations. In practice, this approach is favorably combined with an isotope dilution method which enhances the reliability of measurements at low substrate concentrations. The usefulness of the theoretical and practical approach is demonstrated in investigations of HMG-CoA reductase present in membrane fractions isolated from radish seedlings. The enzyme in the two main membrane fractions obtained by centrifugation at 16000 X g ( P16000 ) and at 105000 X g ( P105000 ) appears to be independently regulated by phytochrome and by phytohormones. Whereas active phytochrome decreases the apparent V of HMG-CoA reductase in the P105000 without affecting the Km, it increases the apparent Km in the P16000 . Kinetin treatment also results in a higher apparent Km of the enzyme in the P16000 fraction. Gibberellic acid and indoleacetic acid did not exhibit such a clear effect.

Differential inhibition by mevinolin of prenyllipid accumulation in radish seedlings

We have studied in intact radish seedlings the effects of mevinolin (at concentrations of 0.25 to 5 μᴍ), a specific inhibitor of HMG-CoA reductase, and, therefore, of mevalonate biosynthesis, on the production of various isopentenoids and prenyllipids. Whereas the content of free desmethyl sterols was decreased steadily, only depending on the concentration of inhibitor present in the parts of seedlings investigated separately (e.g. roots, hypocotyls. and cotyledons), the effect on ubiquinone accumulation was different. Irrespective of the part of seedlings being analyzed, the maximal inhibition reached was 50%. Plastidic pigment accumulation, however, as well as that of chloroplast quinones (plastoquinone and phylloquinone), appeared even to be enhanced at low inhibitor concentrations and was not significantly lowered by application of 5 μᴍ mevinolin. α-Tocopherol showed a similar profile in the dose response to compounds known to be exclusively synthesized in the plastid. The results indicate a differential accessibility of the mevalonate synthesizing enzymes presumably present in the cytoplasm, mitochondria and plastids in respect to the inhibitory action of mevinolin. If prenyllipid formation in the different cell compartments solely depended on cytoplasmic mevalonate biosynthesis, all prenyllipids should be affected to the same extent as the sterols, which are exclusively synthesized by cytoplasmic enzymes.

Selected natural and synthetic enzyme inhibitors of sterol biosynthesis as molecular probes for in vivo studies concerning the regulation of plant growth

Abstract Recently it was shown that radish seedlings ( Raphanus sativus L.) treated with the fungal metabolite mevinolin, a specific inhibitor of membrane-bound 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and thus of mevalonate biosynthesis, exhibit a drastical decrease in root elongation growth. This effect appeared to be specifically paralleled by a decrease in free desmethylsterol content (Bach and Lichtenthaler, Physiol. Plant., 59 (1983) 50). Therefore, some selected natural and synthetic compounds, including systemic fungicides or hypocholesterolemic drugs, known or expected to interfere with later membrane-bound enzymes of the pathway leading to phytosterols, were tested upon their ability to induce similar growth reactions. So far, mevinolin appeared to be the most specific growth inhibitor followed by 15-azasterol, another natural antibiotic. Generally the growth reactions of treated seedlings appear to be quite similar. Therefore, the radish root test system described here may be useful as a rapid screening system in the search for biocides interfering with sterol biosynthesis as well as a test system for rationally designed inhibitors of the sterol pathway upon their ability to exhibit an in vivo response in an intact plant system.