SummaryDetoxification of xenobiotic compounds and heavy metals is a pivotal capacity of organisms, in which glutathione (GSH) plays an important role. In plants, electrophilic herbicides are conjugated to the thiol group of GSH, and heavy metal ions form complexes as thiolates with GSH-derived phytochelatins (PCs). In both detoxification processes of plants, phytochelatin synthase (PCS) emerges as a key player. The enzyme is activated by heavy metal ions and catalyzes PC formation from GSH by transferring glutamylcysteinyl residues (c-EC) onto GSH. In this study with Arabidopsis, we show that PCS plays a role in the plant-specific catabolism of glutathione conjugates (GS-conjugates). In contrast to animals, breakdown of GS-conjugates in plants can be initiated by cleavage of the carboxyterminal glycine residue that leads to the generation of the corresponding c-EC-conjugate. We used the xenobiotic bimane in order to follow GS-conjugate turnover. Functional knockout of the two PCS of Arabidopsis, AtPCS1 and AtPCS2, revealed that AtPCS1 provides a major activity responsible for conversion of the fluorescent bimane-GS-conjugate (GS-bimane) into c-ECbimane. AtPCS1 deficiency resulted in a c-EC-bimane deficiency. Transfection of PCS-deficient cells with AtPCS1 recovered c-EC-bimane levels. The level of the c-EC-bimane conjugate was enhanced several-fold in the presence of Cd 2þ ions in the wild type, but not in the PCS-deficient double mutant, consistent with a PCScatalyzed GS-conjugate turnover. Thus AtPCS1 has two cellular functions: mediating both heavy metal tolerance and GS-conjugate degradation.

Ever since plants began to conquer the terrestrial environment, a simple but effective evolutionary strategy has been employed to cope with the combined necessities of preventing an excessive loss of water via the aerial surface while also supporting the vital exchange of CO(2) and O(2) for photosynthesis and respiration. Large areas of the primary above-ground surface of plants are covered by a hydrophobic, non-cellular cuticle which effectively minimizes evaporation and very strongly reduces exchange of CO(2) and O(2). Hence, gas exchange is controlled by regulating stomatal apertures. Upon wounding or entering into secondary growth, however, the epidermis, cuticle, and stomata are replaced by a phellem (cork), which is produced by a lateral cambium, the phellogen. Former stomata are replaced by lenticels, which are multicellular structures and functionally analogous to stomata. In the secondary plant body, phellems effectively prevent the loss of water from the cortex of the stem while lenticels support the exchange of vital gases such as CO(2), O(2), and water vapour. The permeance of these gases via the lenticels reaches a maximum during July and is minimal during autumn and winter. In contrast to stomatal control, gas exchange through phellems is regulated by long-term structural changes. The permeances of cuticles, phellems, and lenticels are compared and discussed.

The outermost phellems of Abies alba Mill., Acer pseudoplatanus L., Aesculus hippocastanum L., Betula potaninii L.C. Hue and Sambucus nigra L. have been isolated enzymatically, resulting in membranes with five to seven heavily suberized cork cell layers. Water and oxygen permeances were determined for the phellem areas without lenticels. A special diaphragm made it possible to quantify permeances of single lenticels for the first time. The water permeance of phellems was in the range of 3x10(-5) to 9x10(-5) ms(-1) and can be predicted from the density of the phellem membranes with 93% accuracy. Embedded waxes amounted to 3% ( Aesculus) and up to 35% ( Betula) of the dry weight but affected water permeance only to a small degree. The sorption isotherms describing the water content of the phellems in relation to relative humidities followed a hyperbolic shape and indicated varying water contents among plant species. It is argued that water transfer across the phellems occurs via the middle lamellae. Phellem membranes were impermeable to oxygen. Removal of the waxes hardly changed this situation. Single lenticels from Betula and Sambucus were significantly more permeable to water and oxygen than phellem areas without lenticels. The water permeance was elevated by factors of 39 for Betula and 12 for Sambucus, the oxygen permeance by factors of 1,202 for Betula and 53 for Sambucus. Extraction of lenticels did not affect permeance. A quantitative comparison of the gas-exchange capacity of lenticels and stomata demonstrated the superiority of stomata. However, differences may be not more than one order of magnitude.

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Sl'MM.AH't''I'he Hux of ozone to plant surfiiccs results in its decompo.sition at the .surface and its uptiike through the cuticle. Ozone deposition \el<)cit\' and ozone permeance were determined for a \-ariety of isolated plant cuticles. Ozone deposition veloeity was lower than determined with whole plants kept in darkness. It declined continuously during exposure to the gas and showed a 'recovery' effect after an interruption of the fumigation. It increased with the moisture content of the cuticles and decreased when the ozone concentration in the surrounding air was raised. The deposition velocity was much higher than cuticular ozone permeance at equivalent ozone concentrations. Due to the ozone decay in the cuticle, ozone permeance was much lower in thick tlian in thin cuticles. E\-en with the most permeable cuticles, ozone uptake under natural conditions is smaller than the Hux through open stomata hy a factor of at least 10 000.

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Glutathionylation of compounds is an important reaction in the detoxification of electrophilic xenobiotics and in the biosynthesis of endogenous molecules. The glutathione conjugates (GS conjugates) are further processed by peptidic cleavage reactions. In animals and plants, g-glutamyl transpeptidases initiate the turnover by removal of the glutamate residue from the conjugate. Plants have a second route leading to the formation of g-glutamylcysteinyl (g-GluCys) conjugates. Phytochelatin synthase (PCS) is well known to mediate the synthesis of heavy metal-binding phytochelatins. In addition, the enzyme is also able to catabolize GS conjugates to the g-GluCys derivative. In this study, we addressed the cellular compartmentalization of PCS and its role in the plant-specific g-GluCys conjugate pathway in Arabidopsis (Arabidopsis thaliana). Localization studies of both Arabidopsis PCS revealed a ubiquitous presence of AtPCS1 in Arabidopsis seedlings, while AtPCS2 was only detected in the root tip. A functional AtPCS1:eGFP (enhanced green fluorescent protein) fusion protein was localized to the cytosolic compartment. Inhibition of the vacuolar import of GS-bimane conjugate via azide treatment resulted in both a strong accumulation of g-GluCys-bimane and a massive increase of the cellular cysteine to GS-bimane ratio, which was not observed in PCS-deficient lines. These findings support a cytosolic action of PCS. Analysis of a triple mutant deficient in both Arabidopsis PCS and vacuolar g-glutamyl transpeptidase GGT4 is consistent with earlier observations of an efficient sequestration of GS conjugates into the vacuole and the requirement of GGT4 for their turnover. Hence, PCS contributes specifically to the cytosolic turnover of GS conjugates, and AtPCS1 plays the prominent role. We discuss a potential function of PCS in the cytosolic turnover of GS conjugates.

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