Robert L. Heath

A new sensitive assay for the measurement of hydroperoxides

The enzyme glutathione (GSH) peroxidase can be used to measure hydroperoxides quantitatively, easily, and specifically. A timed reaction of GSH peroxidase, coupled with the oxidation of NADPH by GSH reductase, allows a direct spectrophotometric measurement of hydroperoxide. Addition of catalase prior to the addition of GSH peroxidase permits the distinction between hydrogen peroxide and organic hydroperoxides. The solvents that can be used with the assay include methanol, ethanol, water, and aqueous solutions of detergents such as Brij 35, Triton X-100, and cetyl trimethyl ammonium bromide. The utility of the method is demonstrated by the measurement of hydrogen peroxide and organic hydroperoxides formed upon ozonolysis of an unsaturated fatty acid.

Possible mechanisms for the inhibition of photosynthesis by ozone.

Tropospheric ozone produced by industrial civilization is widespread. Although the levels are not clearly life threatening, they do have the potential to inhibit normal plant productivity, thought to be by an inhibition of photosynthesis. While the mechanism for this inhibition is not yet clear, there are several hypotheses for its cause. It is unlikely that ozone can penetrate the cell membrane unreacted; therefore, reactions at the plasma membrane either causing general ionic and metabolic disturbance within the cell or causing the production of unidentified toxic products must ultimately produce the alterations within the chloroplasts. While model systems, such as individual biochemicals, isolated chloroplasts, and algae, can give some understanding of possible reactions, they cannot provide the full story. One continuing controversy revolves about the role of stomata in the inhibition process-they play an important role, but the full interaction between stomatal closure and inhibition of photosynthesis has not yet emerged. In order to reach a political compromise on air quality standards, we need to have a good understanding of the fundamental mechanisms by which ozone causes any decline in plant productivity.

Modification of the biochemical pathways of plants induced by ozone: What are the varied routes to change?

When plants are observed under a low dose of ozone, some physiological and metabolic shifts occur. Barring extreme injury such as tissue damage or stomata closure, most of these disruptive changes are likely to have been initiated at the level of gene expression. The belief is oxidative products formed in ozone exposed leaves, e.g. hydrogen peroxide, are responsible for much of the biochemical adjustments. The first line of defense is a range of antioxidants, such as ascorbate and glutathione, but if this defense is overwhelmed, subsequent actions occur, similar to systemic acquired resistance or general wounding. Yet there are seemingly unrelated metabolic responses which are also triggered, such as early senescence. We discuss here the current understanding of gene control and signal transduction/control in order to increase our comprehension of how ozone alters the basic metabolism of plants and how plants counteract or cope with ozone.

The biochemistry of ozone attack on the plasma membrane of plant cells

Substantial research effort is currently being undertaken towards assessing the impact of oxidant air pollutants (generally, ozone) on economically important agricultural crops.1 Many of these studies rely on vast numbers of field trials in order to determine the minimum level of ozone which affects agricultural productivity. While these data provide regulatory agencies with numbers to set air quality standards, these investigations generate little predictive information. Too many variables of the field and crops prevent complete understanding. Air pollution studies need predictive models but we are painfully short of them. This paper will address the initial site of ozone injury to cells in the hope that the process of model building can begin.

Biochemical Mechanisms of Pollutant Stress

A discussion of biochemical mechanisms in this symposium is critical if one wishes to develop a concept of how O3 exposure induces alterations in a plant’s metabolism, which ultimately lower a plant’s final yield or market value. Furthermore, an understanding of what is and is not known will allow future research to be formulated. In keeping with this concept, I hope to describe several possible scenarios for the development of yield reduction due to primary and secondary biochemical responses. I will concentrate upon O3, as it was the major oxidant investigated within the NCLAN studies.

The Modification of Photosynthetic Capacity Induced by Ozone Exposure

Man’s activities continue to increase the level of ozone in the atmosphere. In the absence of strict controls, plants will be forced to grow in a polluted atmosphere. In crops, this higher level of ozone induces a lowering of productivity, while in natural ecosystems the plants become more at risk to other stresses including freezing, pathogens and insects. Knowing the mechanisms by which ozone induces these effects can allow for breeding and horticultural practices that may minimize any injuries or losses. The entry of ozone is through the stomata, so control of the aperture becomes critical to excluding the oxidant. Once inside the tissue, the primary events seem to be within the cell wall which induce changes in membrane permeability and transport in addition to generating toxic compounds. Carbon assimilation is inhibited by ozone exposure, but the question remains of how. A controversy exists about the role of stomatal closure in limiting photosynthesis rather than any other event within the chloroplasts. However, carbon fixation is altered, under some circumstances, by a decline in ribulose 1,5-bisphosphate carboxylase/oxygenase. This decline is induced principally in more mature leaves by a decline in the messenger RNAs for the subunits of the enzyme. Confounding these events is a strong wounding response induced by ozone that includes the production of ethylene, which in turn can interfere with photosynthesis. Placing the multiplicity of events into any coherent scheme is currently very difficult, but research is slowly sorting out these processes.

Age dependent changes in phospholipids and galactolipids in primary bean leaves (Phaseolus vulgaris)

Abstract Primary leaves of Phaseolus vulgaris show concomitant changes in phospholipid, galactolipid, chlorophyll and fresh weight during leaf development from 3 to 32 days after planting. Phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol show only small changes on a mole per cent lipid phosphate basis during leaf development. The chloroplast lipids, phosphatidyl glycerol, monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) all show marked increases and decreases which are coincident with chloroplast development. The decline in the leaf content of chloroplast polar lipids and chlorophyll become evident upon reaching maximal leaf size. The molar ratio of galactolipids (MGDG/DGDG), reaches a maximum value of 2.3 in expanding leaves, but steadily declines during senescence to a minimum value of 1.5 at abscission. The declining ratio is caused by a preferential loss of MGDG in the senescing leaves.

The Energy State and Structure of Isolated Chloroplasts: The Oxidative Reactions Involving the Water-Splitting Step of Photosynthesis

Publisher Summary This chapter focuses on the water oxidation site of photosynthesis and discusses the structural and functional changes in the chloroplasts that control the normal and altered energy transduction pathways. The chapter discusses photosystem II (PS II)—its evolution and kinetics. Photosystems are connected by pools of compounds and interbranching networks of cytochromes, which play important roles in the channeling of free energy throughout the electron transport system. PS II is a locus leading to an oxidized intermediate (Mn-protein), which oxidizes water, thereby providing electrons for photosynthesis and reducing plastoquinone (PQ). The physiological pathways of energy transduction and electron transfer can be altered and inhibited in part by changing the basic structure of the grana. This, in turn, often leads to oxidation-induced deteriorations, which, in the past, have been looked upon as confusing at best or as an uninteresting artifact at worst. The investigation of water splitting is a great endeavor that encompasses many scientific disciplines from physical to biological.

Responses Of Plants To Air Pollutant Oxidants

Publisher Summary This chapter examines the responses of plants to air pollutant oxidants. The oxidants ozone and peroxyacetyl nitrate cause a variety of injury symptoms in plant tissues ranging from excessive water loss, impairment of photosynthetic activity, an imbalance of metabolites, a reduction in cell wall biosynthesis, and cellular collapse and visible necrosis. Visible injury always results in a reduction in growth and in the case of valuable crop plants, a reduction in yield and quality. The chapter presents a hypothesis that oxidants initially alter the selectivity of limiting cellular membranes such that solutes and salts leak from or into organelles and cells. The resulting imbalance of ionic potential and water balance is manifested first by leakage of potassium from the cells followed osmotically by water. This initial loss of membrane differential permeability produces other biochemical effects, which ultimately cause necrosis or cellular collapse and death of the tissue. The chapter discusses the effect of environment on the susceptibility of plants to oxidants and focusses on the role of stomata in regulating ozone uptake.

Breakdown of ozone and formation of hydrogen peroxide in aqueous solutions of amine buffers exposed to ozone

Abstract Aqueous solutions of amine-buffers (e.g., Tris and 2(N-morpholino)ethane-sulfonic acid) removed ozone from a stream of gas as demonstrated by differential spectrophotometry. During the course of this reaction, hydrogen peroxide was formed and the amine group disappeared. Therefore, the use of organic-amine buffers may present a potential problem when used in association with ozone.