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 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.

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 ß-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.

Conjoined mitochondria and plastids in the barley mutant 'albostrians'.

The intercalary meristem and surrounding tissues of the gene induced plastome mutant 'albostrians' of Hordeum vulgare L. were examined in the electron microscope for ultrastructural evidence of membrane continuities between plastids and mitochondria. In well developed tissues the ribosome-deficient plastids were usually in close proximity or appressed to mitochondria of normal appearance. In some sections through the meristemmatic region however the relationship between the two organelles was observed to be of a fused nature. These conjoinings are thought to be similar to those reported in normal living cells using cinephotomicrography but never before observed by transmission electron microscopy.

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

Gibberellic 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.