Publisher Summary This chapter presents detailed information on chlorophylls and carotenoids to give practical directions toward their quantitative isolation and determination in extracts from leaves, chloroplasts, thylakoid particles, and pigment proteins. The chapter focuses on the spectral characteristics and absorption coefficients of chlorophylls, pheophytins, and carotenoids, which are the basis for establishing equations to quantitatively determine them. Therefore, the specific absorption coefficients of the pigments are re-evaluated. This is achieved by using a two-beam spectrophotometer of the new generation, which allows programmed automatic recording and printing out of the proper wavelengths and absorbancy values. Several procedures have been developed for the separation of the photosynthetic pigments, including column (CC), paper (PC), and thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). All chloroplast carotenoids exhibit a typical absorption spectrum that is characterized by three absorption maxima (violaxanthin, neoxanthin) or two maxima with one shoulder (lutein and β-carotene) in the blue spectral region.

Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

REACTIVE OXYGEN SPECIES: Metabolism, Oxidative Stress, and Signal Transduction

http://paymanhassibi.persiangig.com/document/mittler%20oxidative.pdf

Oxidative stress, antioxidants and stress tolerance

Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants

To cope with environmental fluctuations and to prevent invasion by pathogens, plant metabolism must be flexible and dynamic. Active oxygen species, whose formation is accelerated under stress conditions, must be rapidly processed if oxidative damage is to be averted. The lifetime of active oxygen species within the cellular environment is determined by the antioxidative system, which provides crucial protection against oxidative damage. The antioxidative system comprises numerous enzymes and compounds of low molecular weight. While research into the former has benefited greatly from advances in molecular technology, the pathways by which the latter are synthesized have received comparatively little attention. The present review emphasizes the roles of ascorbate and glutathione in plant metabolism and stress tolerance. We provide a detailed account of current knowledge of the biosynthesis, compartmentation, and transport of these two important antioxidants, with emphasis on the unique insights and advances gained by molecular exploration.

ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control

Various equations for the determinations of total chlorophyll and individual amounts of chlorophylls a and b in extracts from plant tissues exist (see Holden, 1976) and some of them (e.g. Arnon, 1949) are widely used. Additional modifications to the equations have also been developed so as to permit an estimate of total carotenoids to be made from the spectrum of the same mixture in diethyl ether (Ziegler & Egle, 1965; Gaudillire, 1974). During the course of studies that involved the use of various solvents we noted large discrepancies (>40%) between estimations made using the different published equations for particular solvents, all of which were known to contain the same amount of pigments. Taking advantage of a t.1.c. method (Lichtenthaler 8t Pfister, 1978) that permits a distinct separation of the two chlorophylls and also the major carotenoids using light petroleum (b.p. 40-6O0C)/dioxane/propan-2-ol (7 :3 : 1, by vol.) as developing solvent, fresh samples of chlorophyll a and b uncontaminated with each other were readily available for re-evaluation of the published specific absorption coefficients. Those values published by Smith & Benitez (1955), using diethyl ether, were found to be still the most acceptable, and relative specific absorption coefficients to these values were established (Table 1) for various other solvents. The red peak maxima of the chlorophylls were shifted to longer wavelengths with increasing polarity of the solvents; in our case diethyl ether, acetone, 80% (v/v) acetone, 96% (v/v) ethanol and methanol. The red absorption peaks of the chlorophylls were also broadened in the same sequence and the values for the specific absorption coefficients decreased. At the same time suitable values for total carotenoids at 470nm were also determined. On the basis of these coefficients the following equations were derived to determine the individual levels of both chlorophyll a (C,) and chlorophyll b (C,) and the total amounts of carotenoids (Cx+c) and chlorophylls (C,+ C,) [in pg.(ml of plant extract)-'] the measured absorbance values (A) at different wavelengths:

Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents

The Spectral Determination of Chlorophylls a and b, as well as Total Carotenoids, Using Various Solvents with Spectrophotometers of Different Resolution*

Various equations for the determination of total chlorophyll and individual amounts of chlorophylls a and b in extracts from plant tissues exist (see Holden 1976) and some of them (e.g. Arnon 1949) have been widely used. Additional modifications to the equations have also been developed so as to permit an estimate of total carotenoids to be made from the spectrum of the same mixture in di-ethyl ether (Ziegler, Egle 1965; Gaudillere 1974).

Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents

Glutathione (GSH), a major antioxidant in most aerobic organisms, is perceived to be particularly important in plant chloroplasts because it helps to protect the photosynthetic apparatus from oxidative damage. In transgenic tobacco plants overexpressing a chloroplast-targeted gamma-glutamylcysteine synthetase (gamma-ECS), foliar levels of GSH were raised threefold. Paradoxically, increased GSH biosynthetic capacity in the chloroplast resulted in greatly enhanced oxidative stress, which was manifested as light intensity-dependent chlorosis or necrosis. This phenotype was associated with foliar pools of both GSH and gamma-glutamylcysteine (the immediate precursor to GSH) being in a more oxidized state. Further manipulations of both the content and redox state of the foliar thiol pools were achieved using hybrid transgenic plants with enhanced glutathione synthetase or glutathione reductase activity in addition to elevated levels of gamma-ECS. Given the results of these experiments, we suggest that gamma-ECS-transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.

http://www.plantcell.org/content/plantcell/11/7/1277.full.pdf

Alan R. Wellburn

Papers

Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents

The Spectral Determination of Chlorophylls a and b, as well as Total Carotenoids, Using Various Solvents with Spectrophotometers of Different Resolution*

Formulae and Program to Determine Total Carotenoids and Chlorophylls A and B of Leaf Extracts in Different Solvents

Elevated Glutathione Biosynthetic Capacity in the Chloroplasts of Transgenic Tobacco Plants Paradoxically Causes Increased Oxidative Stress

Elevated Glutathione Biosynthetic Capacity in the Chloroplasts of Transgenic Tobacco Plants Paradoxically Causes Increased