A. R. Wellburn

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

Summary Specific absorption (α) coefficients for individual carotenoids and chlorophylls a and b , as well as the E 1% 1cm values for combined carotenoids, have been (re)estimated using 6 solvents (80 % acetone, chloroform, diethyl ether, dimethyl formamide, dimethyl sulphoxide, and methanol) using 2 different types of spectrophotometer (0.1—0.5 nm and 1—4 nm band pass resolution). From these values, 2 sets of equations to calculate concentrations of chlorophyll a (C a ), chlorophyll b (C b ) and total carotenoids (C x+c ) in γg mL -1 for the different instrument types were freshly derived or confirmed from earlier publications. These were then tested with 3 different types of spectrophotometers (the two variable types plus 2 nm fixed resolution diode array) using equal aliquots of a mixed extract in the 6 different solvents. These showed that the concentrations and ratios derived by the 2 sets of equations were comparable when used with their own type of spectrophotometer but less so if the inappropriate equations were used. Measurements taken with the diode array spectrophotometer, however, did not give accurate concentrations or ratios of chlorophylls and carotenoids.

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

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 γ-glutamylcysteine synthetase (γ-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 γ-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 γ-ECS. Given the results of these experiments, we suggest that γ-ECS–transformed plants suffered continuous oxidative damage caused by a failure of the redox-sensing process in the chloroplast.

Effects of SO2 and NO2 polluted air upon the ultrastructure of chloroplasts

Abstract The ultrastructure of mature bean tissue exposed to air polluted by various concentrations of SO 2 or NO 2 at maximum stomatal aperture for short periods was compared with similar unpolluted tissue, using an electron microscope. Both pollutants caused swelling of the thylakoids within the chloroplasts, but no extra-chloroplastidic damage was detected. This thylakoid swelling could be reversed by removing the pollutant from the air flow or changing the rate of gas flow within the tissue. The phenomenon is discussed in relation to previously-reported diminution in the rate of photosynthesis when higher plants are exposed to either SO 2 or NO 2 .

Induction of Ascorbate Peroxidase and Glutathione Reductase Activities by Interactions of Mixtures of Air Pollutants

The response of ascorbate peroxidase and glutathione reductase activities in peas (Pisum sativum var. Waverex) was investigated after three weeks of exposure to mixed fumigations with SO2, NO2 and O3 (0.050 parts per million each) and increasing concentrations of O3 (0-0.150 parts per million). The results show that plants respond similarly to a high concentration (0.150 parts per million) of a single air pollutant (ozone) and to mixtures of air pollutants (SO2, NO2 and O3) when individual concentrations are low (0.050 parts per million each). In both cases, levels of ascorbate peroxidase and glutathione reductase activities were approximately twice those to be found in plants grown in charcoal-filtered air (p less than 0.01).

Synergistic effect of SO2 and NO2 polluted air upon enzyme activity in pea seedlings

Nine day-old seedlings of Pisum sativum (var. Feltham First) were fumigated with sulphur dioxide, nitrogen dioxide, or a combination of both, for six days. A comparison of the activities of ribulose diphosphate carboxylase (RuDPC), glutamate-pyruvate transaminase (GPT), glutamate-oxaloacetate transaminase (GOT) and peroxidase enzymes in extracts from fumigated seedlings showed various differences with respect to those of control seedlings. In some cases the threshold concentration of a pollutant required to produce a change in enzyme activity was lowered when combinations of pollutants were used. A synergistic effect between the pollutants was clearly reflected in the changes in enzyme activity of RuDPC (P < 0·01) and peroxidase (P < 0·001) at concentrations within the ranges 0–2 ppm SO2 and 0–0·1 ppm NO2 and also peroxidase (P < 0·001) at concentrations within the ranges 0–0·2 ppm SO2 and 0–1 ppm NO2.

Influence of gibberellic and abscisic acids and the growth retardant, CCC, upon plastid development

SummaryGibberellic acid (GA3) enhances ultrastructural morphogenesis of plastids in greening cereals whilst abscisic acid (ABA) and CCC have the reverse effect over a shorter period. GA3 application counteracts the ABA and CCC inhibition of membrane development and, over longer periods of greening, the fastest rate of chloroplast development is shown in the presence of both GA3 and ABA. Experiments with isolated etioplasts show that the ABA inhibition of development also occurs and can be counteracted with GA3 treatment but no individual enhancement of plastid morphogenesis by GA3 was detected.Ribulose 1,5-diphosphate carboxylase (RuDPC) acitvity was also increased with applications of GA3 and reduced with ABA treatment. The initial levels of RuDPC activity in the presence of CCC were concentration dependant; high in 10-3 M CCC and low in 10-6 M CCC but activity returned to normal levels after CCC application was stopped. Experiments with isolated etio-chloroplasts gave similar results but levels of RuDPC activity declined rapidly in both illuminated and un-illuminated incubated suspensions of intact etioplasts.

Relationship between foliar injury and changes in antioxidant levels in red and Norway spruce exposed to acidic mists

Elevated levels of total glutathione and enhanced activities of glutathione reductase and ascorbate peroxidase were found in needles of red spruce which had been exposed to acidic mists. Reduced levels of ascorbate were also detected. Such observations suggest that oxidative stress is involved in processes which resist foliar injury caused by acidic misting. Different ionic compositions of the acidic mists applied had pronounced effects on the levels of these antioxidants and the activities of related enzymes. Sulphate was found to be most effective in causing increases in glutathione, while additions of ammonium and/or nitrate mitigated the effects of sulphuric acid. Moreover, it is the composition of ions in the applied mists, rather than the levels of acidity, that determines the extent of the overall response of red spruce. By contrast, although acidic mists caused similar increases in glutathione content of Norway spruce needles, no statistically significant changes of ascorbate or related enzymic activities were found.

Changes in plastid envelope polypeptides during chloroplast development

SummaryA quantitative estimation of sodium dodecyl sulphate-extractable plastid envelope polypeptides during greening of Avena sativa L. laminae is described, combined with protein distribution and plastid number studies over the same period.A primary light-dependent and cytoplasm-dependent increase in both total plastid protein and envelope associated protein during the first 30 minutes of greening was observed, followed by a period during which release of envelope-associated protein either into the plastid or into the cytoplasm, or both, takes place. After 8 hours greening an increase in plastid envelope protein reflects a requirement for an increased plastid surface area during chloroplast maturation.A comparison of the behaviour of envelope membranes during illuminated in vitro incubation of isolated etio-plasts with plastids isolated from tissue given similar illumination pre-treatments, indicates that the early increase in envelope-associated protein is real and requires the participation of the cytoplasm.

Chloroplast development in low light-grown barley seedlings.

Segments of 7-d low light-grown barley laminae cut at 0.5 cm intervals up from the intercalary meristem were examined ultrastructurally and biochemically. The different regions upwards showed the succession of plastid development in light-grown tissues of eoplasts, amyloplasts, amoeboid, immature and mature plastids as described by Whatley (1977). Semi-crystalline bodies were detected in all of them. The eoplast-amyloplast regions are characterised by a greater proportion of mitochondria and high levels of ATP and 3-phosphoglyceric acid, together with low levels of inorganic phosphate conducive to the activation of ADP glucose pyrophosphorylase. The amoeboid and immature plastid regions have higher levels of inhibitory phosphate and starch breakdown may be responsible for the release of metabolites and energy for development. Segments containing amoeboid and immature plastids also have reduced levels of ATP (and 3-phosphoglyceric acid) as photosynthetic components are synthesised. Using ultrastructural assessments of areas of thylakoids, first β-carotene and violaxanthin, followed by chlorophyll a and lutein and, lastly, chlorophyll b are concentrated in the developing lamellar systems of the immature and mature chloroplasts. The formation of additional membraneous material which spreads these pigment systems over a greater thylakoid area within the plastids is the final stage of plastid morphogenesis in low light-grown seedlings.

Effect of SO2 polluted air upon enzyme activity in plants originating from areas with different annual mean atmospheric SO2 concentrations

Abstract Plants of the broad-leaved dock ( rumex obtusifolius ), originating from areas with different annual mean atmospheric SO 2 concentrations, were exposed to charcoal filtered air or 0·2 ppm SO 2 (⋍520 μg SO 2 /m 3 ) for 11 days. Comparisons were made of the levels of activity of ribulose-diphosphate carboxylase (RuDPC), glutamate-pyrurate transaminase (GPT), glutamate-oxaloacetate transaminase (GOT) and peroxidase in leaf extracts from the plants. The effects of the sulphur dioxide fumigation varied, depending on the age of the plant leaves and the area of origin of the plants. The differential response to SO 2 exposure between the plants from the high mean SO 2 area and the low mean SO 2 area suggested that adaptation to the polluted conditions prevailing in the high mean SO 2 area may have occurred in the plants originating from that area.