Reactive oxygen species (ROS), such as O 2 ÿ and H 2 O 2 , play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O 2 ÿ and H 2 O 2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H 2 O 2 -scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H 2 O 2 -scavenging system of Arabidopsis thaliana collapses, H 2 O 2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H 2 O 2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Glutathione plays a pivotal role in protecting plants from environmental stresses, oxidative stress, xenobiotics, and some heavy metals. Arabidopsis plants treated with cadmium or copper responded by increasing transcription of the genes for glutathione synthesis, ␥ -glutamylcysteine synthetase and glutathione synthetase, as well as glutathione reductase. The response was specific for those metals whose toxicity is thought to be mitigated through phytochelatins, and other toxic and nontoxic metals did not alter mRNA levels. Feeding experiments suggested that neither oxidative stress, as results from exposure to H 2 O 2 , nor oxidized or reduced glutathione levels were responsible for activating transcription of these genes. Jasmonic acid also activated the same suite of genes, which suggests that it might be involved in the signal transduction pathway for copper and cadmium. Jasmonic acid treatment increased mRNA levels and the capacity for glutathione synthesis but did not alter the glutathione content in unstressed plants, which supports the idea that the glutathione concentration is controlled at multiple levels. INTRODUCTIONGlutathione (GSH), the tripeptide ␥ -glutamylcysteinylglycine, is the major source of non-protein thiols in most plant cells (Bergmann and Rennenberg, 1993). GSH plays a central role in protecting plants from environmental stresses, including oxidative stress due to the generation of active oxygen species, xenobiotics, and some heavy metals.GSH is involved in quenching reactive oxygen species (Larson, 1988; Alscher, 1989; Foyer et al., 1994b). The ascorbate/GSH cycle reduces H 2 O 2 to water (Foyer and Halliwell, 1976; Alscher, 1989). Ascorbate is also important in maintaining ␣ -tocopherol in the reduced state and therefore links GSH to the dominant free radical scavenger in membranes (Hess, 1994). Plants detoxify many organic contaminants by conjugating them or their metabolites to GSH for storage or further metabolism (Lamoureux et al., 1994). These reactions are catalyzed by glutathione S -transferases (GSTs). Plants are protected from some metals, with cadmium and copper being the most studied, by a group of ␥ -glutamylcysteine ( ␥ -EC) peptides, the phytochelatins (PCs). These molecules have the general structure ( ␥ -Glu-Cys) 2-11 -Gly. They are formed by the polymerization of GSH catalyzed by the transpeptidase phytochelatin synthase (Grill et al., 1985(Grill et al., , 1987 Chen et al., 1997). The PCs bind metals in the cytosol, and the PC metal complex is sequestered in the vacuole (Rauser, 1990).GSH is synthesized from glutamate, cysteine, and glycine by a two-step ATP-dependent reaction (Meister and Anderson, 1983). The first reaction forms ␥ -EC from glutamate and cysteine by the enzyme ␥ -EC synthetase (Hell and Bergmann, 1990), which is encoded by gsh1 (May and Leaver, 1995). GSH is then synthesized by the ligation of ␥ -EC and glycine in the reaction catalyzed by the enzyme GSH synthetase, which is encoded by gsh2 (Wang and Oliver, 1996). When GSH is oxidized as part of ...

Drought is one of the most important environmental constraints limiting plant growth and agricultural productivity. To understand the underlying mechanism of drought tolerance and to identify genes for improving this important trait, we conducted a gain-of-function genetic screen for improved drought tolerance in Arabidopsis thaliana. One mutant with improved drought tolerance was isolated and designated as enhanced drought tolerance1. The mutant has a more extensive root system than the wild type, with deeper roots and more lateral roots, and shows a reduced leaf stomatal density. The mutant had higher levels of abscisic acid and Pro than the wild type and demonstrated an increased resistance to oxidative stress and high levels of superoxide dismutase. Molecular genetic analysis and recapitulation experiments showed that the enhanced drought tolerance is caused by the activated expression of a T-DNA tagged gene that encodes a putative homeodomain-START transcription factor. Moreover, overexpressing the cDNA of the transcription factor in transgenic tobacco also conferred drought tolerance associated with improved root architecture and reduced leaf stomatal density. Therefore, we have revealed functions of the homeodomain-START factor that were gained upon altering its expression pattern by activation tagging and provide a key regulator that may be used to improve drought tolerance in plants.

A functional analysis of the role of glutathione in protecting plants from environmental stress was undertaken by studying Arabidopsis that had been genetically modified to have altered glutathione levels. The steady-state glutathione concentration in Arabidopsis plants was modified by expressing the cDNA for ␥-glutamyl-cysteine synthetase (GSH1) in both the sense and antisense orientation. The resulting plants had glutathione levels that ranged between 3% and 200% of the level in wild-type plants. Arabidopsis plants with low glutathione levels were hypersensitive to Cd due to the limited capacity of these plants to make phytochelatins. Plants with the lowest levels of reduced glutathione (10% of wild type) were sensitive to as little as 5 m Cd, whereas those with 50% wild-type levels required higher Cd concentrations to inhibit growth. Elevating glutathione levels did not increase metal resistance. It is interesting that the plants with low glutathione levels were also less able to accumulate anthocyanins supporting a role for glutathione S-transferases for anthocyanin formation or for the vacuolar localization and therefore accumulation of these compounds. Plants with less than 5% of wild-type glutathione levels were smaller and more sensitive to environmental stress but otherwise grew normally.Glutathione (GSH), the tripeptide ␥-glutamylcysteinyl-Gly, is the major source of non-protein thiols in most plant cells (Bergmann and Rennenberg, 1993). The chemical reactivity of the thiol group of glutathione makes it particularly suitable to serve a broad range of biochemical functions in all organisms. It has an oxidation reduction potential of Ϫ0.23 V that allows it to act as an effective electron acceptor and donor for numerous biological reactions. The nucleophilic nature of the thiol group also is important in the formation of mercaptide bonds with metals and for reacting with select electrophiles. This reactivity, along with the relative stability and high water solubility of GSH, makes it an ideal biochemical to protect plants against stress including oxidative stress, heavy metals, and certain exogenous and endogenous organic chemicals.

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.