Glutathione – linking cell proliferation to oxidative stress

SignificanceThe multifaceted functions of reduced glutathione (gamma-glutamyl–cysteinyl–glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions.Recent advancesThe intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about −300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment.Critical issuesThe concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined.Future directionsThe nuclear GSH pool provides an appropriate redox environment for essential nuclear functions. Future work will focus on how this essential thiol interacts with the nuclear thioredoxin system and nitric oxide to regulate genetic and epigenetic mechanisms. The characterization of redox-regulated cell cycle proteins in plants, and the elucidation of mechanisms that facilitate GSH accumulation in the nucleus are keep steps to unravelling the complexities of nuclear redox controls.     

Nuclear glutathione

Glutathione (GSH) is a linchpin of cellular defences in plants and animals with physiologically-important roles in the protection of cells from biotic and abiotic stresses. Moreover, glutathione participates in numerous metabolic and cell signalling processes including protein synthesis and amino acid transport, DNA repair and the control of cell division and cell suicide programmes. While it is has long been appreciated that cellular glutathione homeostasis is regulated by factors such as synthesis, degradation, transport, and redox turnover, relatively little attention has been paid to the influence of the intracellular partitioning on glutathione and its implications for the regulation of cell functions and signalling. We focus here on the functions of glutathione in the nucleus, particularly in relation to physiological processes such as the cell cycle and cell death. The sequestration of GSH in the nucleus of proliferating animal and plant cells suggests that common redox mechanisms exist for DNA regulation in G1 and mitosis in all eukaryotes. We propose that glutathione acts as “redox sensor” at the onset of DNA synthesis with roles in maintaining the nuclear architecture by providing the appropriate redox environment for the DNA replication and safeguarding DNA integrity. In addition, nuclear GSH may be involved in epigenetic phenomena and in the control of nuclear protein degradation by nuclear proteasome. Moreover, by increasing the nuclear GSH pool and reducing disulfide bonds on nuclear proteins at the onset of cell proliferation, an appropriate redox environment is generated for the stimulation of chromatin decompaction. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma‐glutamyl cycle in plant's adaptation to environment

The existence of a gamma‐glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma‐glutamyl transferase (GGT)‐assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild‐type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low‐molecular weight range compared to WT. The combined iTRAQ and LC‐MS/MS‐based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH‐associated genes showed that disruption of gamma‐glutamyl cycle in ggt1 knockout‐leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma‐glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.     

The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement

AIR9 is a cytoskeleton‐associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9‐like protein, TbAIR9, is also cytoskeleton‐associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re‐positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non‐equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasize differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite.     

Dehydroepiandrosterone augments sensitivity to γ-ray irradiation in human H4 neuroglioma cells through down-regulation of Akt signaling

Dehydroepiandrosterone (DHEA) modulates sensitivity to radiation-induced injury in human neuroglioma cells (H4) through effects on Akt signalling by glutathione (GSH)-dependent redox regulation. Previous treatment of H4 cells with DHEA for 18 h reduced the γ-ray-induced phosphorylation of Akt, activated p21^waf1 synthesis and up-regulated phosphorylation of Rb independent of p53. These reactions were followed by a decrease in cell number and an increase in apoptosis and G₂/M checkpoint arrest. The suppression of phosphorylation of Akt by DHEA was due to regulation of the dephosphorylation by protein phosphatase 2A (PP2A). DHEA up-regulated the expression of γ-glutamylcysteine synthetase, a rate-limiting enzyme of glutathione (GSH) synthesis, and the levels of GSH to maintain PP2A activity. The results suggested that DHEA increases the sensitivity of cells to γ-ray irradiation by inducing apoptosis and cell cycle arrest through GSH-dependent regulation of the reduced form of PP2A to down-regulate the Akt signalling pathway.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

Characterisation of antioxidants in photosynthetic and non‐photosynthetic leaf tissues of variegated Pelargonium zonale plants

Hydrogen peroxide is an important signalling molecule, involved in regulation of numerous metabolic processes in plants. The most important sources of H₂O₂ in photosynthetically active cells are chloroplasts and peroxisomes. Here we employed variegated Pelargonium zonale to characterise and compare enzymatic and non‐enzymatic components of the antioxidative system in autotrophic and heterotrophic leaf tissues at (sub)cellular level under optimal growth conditions. The results revealed that both leaf tissues had specific strategies to regulate H₂O₂ levels. In photosynthetic cells, the redox regulatory system was based on ascorbate, and on the activities of thylakoid‐bound ascorbate peroxidase (tAPX) and catalase. In this leaf tissue, ascorbate was predominantly localised in the nucleus, peroxisomes, plastids and mitochondria. On the other hand, non‐photosynthetic cells contained higher glutathione content, mostly located in mitochondria. The enzymatic antioxidative system in non‐photosynthetic cells relied on the ascorbate–glutathione cycle and both Mn and Cu/Zn superoxide dismutase. Interestingly, higher content of ascorbate and glutathione, and higher activities of APX in the cytosol of non‐photosynthetic leaf cells compared to the photosynthetic ones, suggest the importance of this compartment in H₂O₂ regulation. Together, these results imply different regulation of processes linked with H₂O₂ signalling at subcellular level. Thus, we propose green‐white variegated leaves as an excellent system for examination of redox signal transduction and redox communication between two cell types, autotrophic and heterotrophic, within the same organ.     

CYLD negatively regulates cell‐cycle progression by inactivating HDAC6 and increasing the levels of acetylated tubulin

CYLD is a tumour‐suppressor gene that is mutated in a benign skin tumour syndrome called cylindromatosis. The CYLD gene product is a deubiquitinating enzyme that was shown to regulate cell proliferation, cell survival and inflammatory responses, mainly through inhibiting NF‐κB signalling. Here we show that CYLD controls cell growth and division at the G₁/S‐phase as well as cytokinesis by associating with α‐tubulin and microtubules through its CAP‐Gly domains. Translocation of activated CYLD to the perinuclear region of the cell is achieved by an inhibitory interaction of CYLD with histone deacetylase‐6 (HDAC6) leading to an increase in the levels of acetylated α‐tubulin around the nucleus. This facilitates the interaction of CYLD with Bcl‐3, leading to a significant delay in the G₁‐to‐S‐phase transition. Finally, CYLD also interacts with HDAC6 in the midbody where it regulates the rate of cytokinesis in a deubiquitinase‐independent manner. Altogether these results identify a mechanism by which CYLD regulates cell proliferation at distinct cell‐cycle phases.     

CD151 mediates netrin‐1‐induced angiogenesis through the Src‐FAK‐Paxillin pathway

Crosstalk between the nervous and vascular systems is important during development and in response to injury, and the laminin‐like axonal guidance protein netrin‐1 has been studied for its involvement in angiogenesis and vascular remodelling. In this study, we examined the role of netrin‐1 in angiogenesis and explored the underlying mechanisms. The effect of netrin‐1 on brain tissues and endothelial cells was examined by immunohistochemistry and western blotting in a middle cerebral artery occlusion model and in human umbilical vein endothelial cells. Cell proliferation and cell cycle progression were assessed by the MTT assay and flow cytometry, and the Transwell and tube formation assays were used to examine endothelial cell motility and function. Netrin‐1 up‐regulated CD151 and VEGF concomitant with the activation of focal adhesion kinase (FAK), Src and Paxillin in vitro and in vivo and the induction of cell proliferation, migration and tube formation in vitro. Silencing of CD151 abolished the effects of netrin‐1 on promoting cell migration and tube formation mediated by the activation of FAK/Src signalling. Netrin‐1 promoted angiogenesis in vitro and in vivo by activating the FAK/Src/Paxillin signalling pathway through a mechanism dependent on the expression of the CD151 tetraspanin, suggesting the existence of a netrin‐1/FAK/Src/CD151 signalling axis involved in the modulation of angiogenesis.     

Phosphorylation of p27KIP1 homologs KRP6 and 7 by SNF1‐related protein kinase–1 links plant energy homeostasis and cell proliferation

SNF1‐related protein kinase–1 (SnRK1), the plant kinase homolog of mammalian AMP‐activated protein kinase (AMPK), is a sensor that maintains cellular energy homeostasis via control of anabolism/catabolism balance. AMPK‐dependent phosphorylation of p27^KIP1 affects cell‐cycle progression, autophagy and apoptosis. Here, we show that SnRK1 phosphorylates the Arabidopsis thaliana cyclin‐dependent kinase inhibitor p27^KIP1 homologs AtKRP6 and AtKRP7, thus extending the role of this kinase to regulation of cell‐cycle progression. AtKRP6 and 7 were phosphorylated in vitro by a recombinant activated catalytic subunit of SnRK1 (AtSnRK1α1). Tandem mass spectrometry and site‐specific mutagenesis identified Thr152 and Thr151 as the phosphorylated residues on AtKRP6‐ and AtKRP7, respectively. AtSnRK1 physically interacts with AtKRP6 in the nucleus of transformed BY–2 tobacco protoplasts, but, in contrast to mammals, the AtKRP6 Thr152 phosphorylation state alone did not modify its nuclear localization. Using a heterologous yeast system, consisting of a cdc28 yeast mutant complemented by A. thaliana CDKA;1, cell proliferation was shown to be abolished by AtKRP6^WT and by the non‐phosphorylatable form AtKRP6^T152A, but not by the phosphorylation‐mimetic form AtKRP6^T152D. Moreover, A. thaliana SnRK1α1/KRP6 double over‐expressor plants showed an attenuated AtKRP6‐associated phenotype (strongly serrated leaves and inability to undergo callogenesis). Furthermore, this severe phenotype was not observed in AtKRP6^T152D over‐expressor plants. Overall, these results establish that the energy sensor AtSnRK1 plays a cardinal role in the control of cell proliferation in A. thaliana plants through inhibition of AtKRP6 biological function by phosphorylation.     

Arabidopsis MPK6 is involved in cell division plane control during early root development,and localizes to the pre‐prophase band,phragmoplast, trans‐Golgi network and plasma membrane

The proper spatial and temporal expression and localization of mitogen‐activated protein kinases (MAPKs) is essential for developmental and cellular signalling in all eukaryotes. Here, we analysed expression, subcellular localization and function of MPK6 in roots of Arabidopsis thaliana using wild‐type plants and three mpk6 knock‐out mutant lines. The MPK6 promoter showed two expression maxima in the most apical part of the root meristem and in the root transition zone. This expression pattern was highly consistent with ‘no root’ and ‘short root’ phenotypes, as well as with ectopic cell divisions and aberrant cell division planes, resulting in disordered cell files in the roots of these mpk6 knock‐out mutants. In dividing root cells, MPK6 was localized on the subcellular level to distinct fine spots in the pre‐prophase band and phragmoplast, representing the two most important cytoskeletal structures controlling the cell division plane. By combining subcellular fractionation and microscopic in situ and in vivo co‐localization methods, MPK6 was localized to the plasma membrane (PM) and the trans‐Golgi network (TGN). In summary, these data suggest that MPK6 localizing to mitotic microtubules, secretory TGN vesicles and the PM is involved in cell division plane control and root development in Arabidopsis.     

Triggering the cell cycle in plants

In essence, the mitotic cell cycle in eukaryotes involves the duplication and separation of chromosomes, coupled to the process of dividing one cell into two. Cytokinesis is therefore the culmination of a series of events that were triggered during G1 phase, and brings the daughter cells back to the starting position in G1 for another possible round of division. In all eukaryotes, progression through the cell cycle is controlled by cyclin-dependent kinases that bind to positive regulators called cyclins. This review explores some of the pathways that trigger the plant cell cycle, with emphasis on the G1 phase. Examples include signalling pathways involving glutathione and cellular redox potential, the possible existence of a G1 DNA-damage checkpoint, and the plant hormones auxin and cytokinin. Progress in understanding the link between cell proliferation, cell differentiation and the cell-cycle machinery in a developmental context is discussed.