Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6-to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H 2 O 2 -sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.The complex relationships between programmed cell death (PCD) and natural senescence observed during leaf development are far from understood. However, one clear distinction is that senescence in leaves is essentially reversible, but PCD is not (Thomas et al., 2003). The genetically programmed cell suicide events that comprise PCD are triggered by enhanced levels of reactive oxygen species (ROS; Chen and Dickman, 2004;Laloi et al., 2004;Wagner et al., 2004). However, senescence-enhanced genes are also expressed in response to ROS (Navabpour et al., 2003).While the chemical nature of ROS dictates that they are potentially harmful to cells, plants use ROS as second messengers in signal transduction cascades regulating diverse processes such as mitosis, tropisms, and cell death. It is now well accepted that ROS accumulation is crucial to plant development as well as defense (Foyer and Noctor, 2005a). ROS signal transduction will ensue only if ROS escape destruction by cellular antioxidants that determine the lifetime and specificity of the signal. Ascorbic acid (AA) and glutathione are the major redox buffers of the plant cells, and they themselves are also signal-transducing molecules that can either signal independently or further transmit ROS signals (Fig. 1). They are thus intrinsic to redox homeostasis and redox-signaling events (Foyer and Noctor, 2005b).ROS production is often genetically programmed, for example, during the hypersen...

SummaryA functional cloning approach using the oxidant-sensitive yeast mutant, Dyap1, was employed to identify plant genes involved in tolerance of oxidative stress. In this screen, we identified an Arabidopsis late embryogenesisabundant (LEA)-like protein, AtLEA5, which increased the tolerance of Dyap1 cells to the oxidants H 2 O 2 , diamide, menadione and tert-butyl hydroperoxide. Unlike canonical LEAs, AtLEA5 is constitutively expressed in roots and reproductive organs but not in seeds. In leaves of short-day grown plants, AtLEA5 transcripts exhibited a diurnal pattern of regulation, where transcripts were repressed in the light and abundant in the dark. Expression of AtLEA5 in leaves was induced by oxidants, ABA and dehydration. Use of abi1-1 (ABAinsensitive) and aba1-1 (ABA-deficient) Arabidopsis mutants indicated that drought induction of AtLEA5 required ABA synthesis but was independent of the ABI1 gene product. Abscisic acid and H 2 O 2 induction of AtLEA5 was also independent of the OXI1 protein kinase. Constitutive overexpression of AtLEA5 resulted in increased root growth and shoot biomass, both in optimal conditions and under H 2 O 2 stress. However, in comparison with wild type, photosynthesis in overexpressing plants was more susceptible to drought. These features suggest that AtLEA5 has a unique function among LEA proteins in that it plays a specific role in protection against oxidative stress involving decreased photosynthesis. This protein functions as part of a complex network of defences that contribute to robustness of plants under stress by minimizing the negative effects of oxidation.

The T-box transcription factors TBX2 and TBX3 are overexpressed in several cancers and are able to bypass senescence by repressing ARF and p21 WAF1/CIP1/SDII . Although these studies suggest that they may both contribute to the oncogenic process by repressing common targets, whether they have redundant or distinct roles in cancers where they are both overexpressed remains to be elucidated. Importantly, when Tbx2 function is inhibited in melanoma cells lacking Tbx3, the cells senesce, but whether this is possible in melanoma cells overexpressing both proteins is not known. An understanding of this issue may have important implications for the design of an effective pro-senescence therapy. In this study, the authors used a sh-RNA approach to knock down TBX2 and TBX3 individually in 2 human melanoma cell lines that overexpress both these factors and then examined their specific involvement in the oncogenic process. They demonstrate, using in vitro and in vivo cell proliferation, as well as colony-and tumor-forming ability and cell motility assays, that TBX2 and TBX3 have distinct roles in melanoma progression. In the tested lines, although TBX2 could promote proliferation and transformation and was required by primary melanoma cells for immortality, TBX3 was required for tumor formation and cell migration. These findings were reproducible in a human breast cancer cell line, which confirms that TBX2 and TBX3, although highly homologous, do not have redundant roles in the transformation process of cancers where they are both overexpressed. These results have important implications for the development of new cancer treatments and in particular for melanoma, which is a highly aggressive and intractable cancer.

SAG21/AtLEA5 belongs to the late embryogenesisassociated (LEA) protein family. Although it has been implicated in growth and redox responses, its precise roles remain obscure. To address this problem, we characterized root and shoot development and response to biotic stress in SAG21/AtLEA5 over-expressor (OEX) and antisense (AS) lines. AS lines exhibited earlier flowering and senescence and reduced shoot biomass. Primary root length was reduced in AS lines, as was the number of laterals relative to the primary root. Root hair number was unchanged but root hair length was proportional to SAG21/AtLEA5 expression level, with longer root hairs in OEX lines and shorter root hairs in AS, relative to wild type. Growth of the fungal nectroph, Botrytis cinerea and of a virulent bacterial pathogen (Pseudomonas syringae pv. tomato) was affected by SAG21/AtLEA5 expression; however, growth of an avirulent P.syringae strain was unaffected. A SAG21/ AtLEA5-YFP fusion was localized to mitochondria, raising the intriguing possibility that SAG21 interacts with proteins involved in mitochondrial ROS signalling, which in turn, impacts on root development and pathogen responses.

A cDNA corresponding to 1-Cys peroxiredoxin, an evolutionarily conserved thiol-specific antioxidant enzyme, was isolated from Xerophyta viscosa Baker, a resurrection plant indigenous to Southern Africa and belonging to the family Velloziaceae. The cDNA, designated XvPer1, contains an open reading frame that encodes a polypeptide of 219 residues with a predicted molecular weight of 24.2 kDa. The XvPer1 polypeptide shows significant sequence identity (approx. 70%) to other recently identified plant 1-Cys peroxiredoxins and relatively high levels of sequence similarity (approx. 40%) to non-plant 1-Cys peroxiredoxins. The XvPer1 cDNA contains a putative polyadenylation site. As for all 1-Cys peroxiredoxins identified to date, the amino acid sequence proposed to constitute the active site of the enzyme, PVCTTE, is highly conserved in XvPer1. It also contains a putative bipartite nuclear localization signal. Southern blot analysis revealed that there is a single copy of XvPer1 in the X. viscosa genome. All angiosperm 1-Cys peroxiredoxins described to date are seed-specific and absent in vegetative tissues even under stress conditions; therefore, XvPer1 is unique in that it is expressed in the vegetative tissues of X. viscosa. The XvPer1 transcript was absent in fully hydrated X. viscosa tissue but levels increased in tissues subjected to abiotic stresses such as dehydration, heat (42 degrees C), high light intensity (1,500 micro mol photons m(-2) s(-1)) and when treated with abscisic acid (100 micro M ABA) and sodium chloride (100 mM NaCl). Western blot analyses correlated with the patterns of expression of XvPer1 transcripts under different stress conditions. Immunofluorescence analyses revealed that XvPer1 is localized in the nucleus of dehydrated X. viscosa leaf cells. These results suggest that XvPer1 is a stress-inducible gene, which may function to protect nucleic acids within the nucleus against oxidative injury.

Aims To report on molecular mechanisms of foetal haemoglobin (HbF) induction by hydroxyurea (HU) for the treatment of Sickle Cell Disease (SCD). Study Design Systematic review. Results Studies have provided consistent associations between genomic variations in HbF-promoting loci and variable HbF level in response to HU. Numerous signal transduction pathways have been implicated, through the identification of key genomic variants in BCL11A, HBS1L-MYB, SAR1 or XmnI polymorphism that predispose the response to the treatment, and signal transduction pathways, that modulate γ-globin expression (cAMP/cGMP; Giα/JNK/Jun; methylation and microRNA). Three main molecular pathways have been reported: 1) Epigenetic modifications, transcriptional events and signalling pathways involved in HU-mediated response, 2) Signalling pathways involving HU-mediated response and 3) Post-transcriptional pathways (regulation by microRNAs). Conclusions The complete picture of HU-mediated mechanisms of HbF production in SCD remains elusive. Research on post-transcriptional mechanisms could lead to therapeutic targets that may minimize alterations to the cellular transcriptome.

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclindependent kinase inhibitors p19 ARF and p21 WAF1/CIP1/SDII. Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using sitedirected mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21 WAF1/CIP1/SDII promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.

The AKT3 signalling pathway plays a critical role in melanoma formation and invasion and components of this signalling cascade are therefore attractive targets for the treatment of malignant melanoma. Recent evidence show that the embryonically important TBX3 transcription factor is upregulated in a subset of melanomas and plays a key role in promoting melanoma formation and invasion, in part by repressing the cell adhesion molecule E-cadherin. We have identified TBX3 as a key substrate of AKT3 in melanomagenesis. Briefly, using site-directed mutagenesis and in vitro kinase assays, we have identified the AKT3 target site at serine residue 720 in the TBX3 protein and show that this site is phosphorylated in vivo. Importantly, we show by western blotting, immunofluorescence, reporter, migration and invasion assays that the phosphorylation at S720 promotes TBX3 protein stability, nuclear localization, transcriptional repression of E-cadherin, and its role in cell migration and invasion. Our results identify a novel signalling and transcriptional network linking AKT3, TBX3 and E-cadherin during melanoma migration and invasion and reveals TBX3 as a potential target for anti-metastatic therapeutics.