Glutathione has numerous roles in cellular defence and in sulphur metabolism. These functions depend or impact on the concentration and/or redox state of leaf glutathione pools. Effective function requires homeostatic control of concentration and redox state, with departures from homeostasis acting as signals that trigger adaptive responses. Intercellular and intracellular glutathione pools are linked by transport across membranes. It is shown that glutathione can cross the chloroplast envelope at rates similar to the speed of biosynthesis. Control of glutathione concentration and redox state is therefore due to a complex interplay between biosynthesis, utilization, degradation, oxidation/reduction, and transport. All these factors must be considered in order to evaluate the significance of glutathione as a signalling component during development, abiotic stress, or pathogen attack.

SUMMARYTrehalose and associated metabolites are part of the sugar signalling system in plants and have profound effects on development. Disruption of the TREHALOSE 6-PHOSPHATE SYNTHASE (TPS1) gene in Arabidopsis results in delayed embryo growth, altered cell wall morphology and carbon metabolism and abortion at the torpedo stage. Here we investigate the role of the TPS1 gene in post-embryonic development using two approaches. In the first we use the seed-specific ABI3 promoter to drive the TPS1 cDNA during embryo development, resulting in rescue of the embryo-lethal tps1 phenotype. Lack of expression from the ABI3::TPS1 transgene in post-germinative tps1 seedlings results in severe growth arrest, accumulation of soluble sugars and starch and leads to an increase in expression of genes related to ABA signalling. In the second approach we use TILLING (targeted induced local lesions in genomes) to generate three weaker, non-embryo-lethal, alleles (tps1-11, tps1-12 and tps1-13) and use these to demonstrate that the TPS1 protein plays a key role in modulating trehalose 6-phosphate (T6P) levels in vegetative tissues of Arabidopsis. All three weaker alleles give a consistent phenotype of slow growth and delayed flowering. Germination of tps1-11, tps1-12 and tps1-13 is hypersensitive to ABA with the degree of hypersensitivity correlating with the decrease in T6P levels in the different alleles. Stomatal pore aperture is regulated by ABA, and this was found to be affected in tps1-12. Our results show that the TPS1 gene product plays an essential role in regulating the growth of vegetative as well as embryogenic tissue in a mechanism involving ABA and sugar metabolism.

SummaryThe tps1 mutant, which is disrupted in the TREHALOSE-6-PHOSPHATE SYNTHASE 1 gene, has been previously characterized as a recessive embryo lethal. tps1 embryos do not develop past late torpedo or early cotyledon stage. We report here that at the ultrastructural, biochemical, and transcriptional levels tps1 exhibits many features typically associated with the maturation phase. The appearance of storage reserve transcripts and organelles follows the same temporal pattern in tps1 and wild-type (WT) embryos in the same silique as does accumulation of storage lipid and protein. The mutant plastids accumulate large starch granules that persist until the end of seed development, in contrast with WT plastids where starch accumulation is transient. The transcriptome of tps1 embryos shows a coordinate downregulation of genes involved in starch and sucrose degradation. Interestingly, genes involved in lipid mobilization and gluconeogenesis are induced in tps1 embryos. The cell walls of tps1 embryos show a remarkable degree of thickening at the ultrastructural level and immunodetection of cell wall components shows that altered deposition of pectins accounts for this altered morphology. Consistent with this at the transcriptome level, genes involved in sugar nucleotide and pectin metabolism are altered in the mutant. The frequency of cell division in tps1 embryos is half that of the wild type at the heart and torpedo stages. These results suggest that TPS1 may play a major role in coordinating cell wall biosynthesis and cell division with cellular metabolism during embryo development.

1366I.1366II.1367III.1368IV.1368V.1369VI.1370VII.1372VIII.1372IX.1376X.13771377References1377 Summary The aim of producing sustainable liquid biofuels and chemicals from lignocellulosic biomass remains high on the sustainability agenda, but is challenged by the costs of producing fermentable sugars from these materials. Sugars from plant biomass can be fermented to alcohols or even alkanes, creating a liquid fuel in which carbon released on combustion is balanced by its photosynthetic capture. Large amounts of sugar are present in the woody, nonfood parts of crops and could be used for fuel production without compromising global food security. However, the sugar in woody biomass is locked up in the complex and recalcitrant lignocellulosic plant cell wall, making it difficult and expensive to extract. In this paper, we review what is known about the major polymeric components of woody plant biomass, with an emphasis on the molecular interactions that contribute to its recalcitrance to enzymatic digestion. In addition, we review the extensive research that has been carried out in order to understand and reduce lignocellulose recalcitrance and enable more cost‐effective production of fuel from woody plant biomass.

To investigate the intercellular control of glutathione synthesis and its influence on leaf redox state in response to short-term chilling, genes encoding g-glutamylcysteine synthetase (g-ECS) and glutathione synthetase (GSH-S) were cloned from maize (Zea mays) and specific antibodies produced. These tools were used to provide the first information on the intercellular distribution of g-ECS and GSH-S transcript and protein in maize leaves, in both optimal conditions and chilling stress. A 2-d exposure to low growth temperatures (chill) had no effect on leaf phenotype, whereas return to optimal temperatures (recovery) caused extensive leaf bleaching. The chill did not affect total leaf GSH-S transcripts but strongly induced g-ECS mRNA, an effect reversed during recovery. The chilling-induced increase in g-ECS transcripts was not accompanied by enhanced total leaf g-ECS protein or extractable activity. In situ hybridization and immunolocalization of leaf sections showed that g-ECS and GSH-S transcripts and proteins were found in both the bundle sheath (BS) and the mesophyll cells under optimal conditions. Chilling increased g-ECS transcript and protein in the BS but not in the mesophyll cells. Increased BS g-ECS was correlated with a 2-fold increase in both leaf Cys and g-glutamylcysteine, but leaf total glutathione significantly increased only in the recovery period, when the reduced glutathione to glutathione disulfide ratio decreased 3-fold. Thus, while there was a specific increase in the potential contribution of the BS cells to glutathione synthesis during chilling, it did not result in enhanced leaf glutathione accumulation at low temperatures. Return to optimal temperatures allowed glutathione to increase, particularly glutathione disulfide, and this was associated with leaf chlorosis.

The acclimation of reduced glutathione (GSH) biosynthesis and GSH-utilizing enzymes to salt stress was studied in two tomato species that di¡er in stress tolerance. Salt increased GSH content and GSH:GSSG (oxidized glutathione) ratio in oxidative stress-tolerant Lycopersicon pennellii (Lpa) but not in Lycopersicon esculentum (Lem). These changes were associated with salt-induced upregulation of Q Q-glutamylcysteine synthetase protein, an e¡ect which was prevented by preincubation with buthionine sulfoximine. Salt treatment induced glutathione peroxidase and glutathione-S-transferase but not glutathione reductase activities in Lpa. These results suggest a mechanism of coordinate upregulation of synthesis and metabolism of GSH in Lpa, that is absent from Lem.

Changes in CuZn-SOD activity and content in isolated wheat chloroplasts under the light, and the involvement of protease(s) and/or active oxygen species in this process were studied. Both SOD activity and content decayed with exposure time to photooxidative stress. Ascorbate, a H2O2 scavenger, prevented photooxidation-associated inactivation of SOD, while benzoate, a .OH scavenger, prevented SOD degradation. Wheat chloroplasts incubated in the dark did not hydrolyze exogenous or endogenous SOD, either H2O2-pretreated or not. Protease inhibitors did not prevent SOD degradation under photooxidative treatment, suggesting that plastid protease(s) did not participate in this process. Purified chloroplast CuZn-SOD was exposed to H2O2 and O2- or .OH-generating systems. O2- had no effect on either SOD activity or stability (estimated by native PAGE). H2O2 up to 700 microM inhibited SOD in a dose-dependent manner and induced charge/mass changes as seen by native PAGE. .OH also reduced SOD activity by inducing its fragmentation. High levels of active oxygen, as can be generated under strong stress conditions, could directly inactivate and degrade chloroplastic SOD.

The participation of the antioxidant system in the drought tolerance of wheat cultivars (Triticum aestivum L.) was studied under field and in vitro conditions. Under field conditions, drought tolerance was evaluated by the capacity to maintain the grain yield under drought, which was higher in cvv. Elite and La Paz than in the sensitive cvv. Oasis and Cruz Alta. Tolerant cultivars showed lower relative water content (RWC) and lower above-ground vegetative biomass than sensitive cultivars. Field assays did not show a clear correlation between water-stress tolerance and antioxidant system behaviour. However, when leaves of cvv. with contrasting drought tolerance were subjected to osmotic stress in vitro, clear differences in the antioxidant system activity and oxidative damage between cvv. were observed. In the tolerant cultivar Elite, it was possible to observe an increase in ascorbate peroxidase (APX), superoxide dismutase (SOD) and glutathione reductase (GR) activities, a higher glutathione (GSH) and ascorbate content and less oxidative damage than in the sensitive cultivar Oasis, which showed no changes or only slight decreases in the enzyme activities. These results indicate that water stress tolerance is in part associated with the antioxidant system activity, and suggest that the behaviour of the antioxidant systemin vitro assays can be used as an early selection tool.