dTMP imbalance through thymidylate 5'-phosphohydrolase activity induces apoptosis in triple-negative breast cancers.

Immunotherapy has a number of advantages over traditional anti-tumor therapy but can cause severe adverse reactions due to an overactive immune system. In contrast, a novel metabolic treatment approach can induce metabolic vulnerability through multiple cancer cell targets. Here, we show a therapeutic effect by inducing nucleotide imbalance and apoptosis in triple negative breast cancer cells (TNBC), by treating with cytosolic thymidylate 5'-phosphohydrolase (CT). We show that a sustained consumption of dTMP by CT could induce dNTP imbalance, leading to apoptosis as tricarboxylic acid cycle intermediates were depleted to mitigate this imbalance. These cytotoxic effects appeared to be different, depending on substrate specificity of the 5' nucleotide or metabolic dependency of the cancer cell lines. Using representative TNBC cell lines, we reveal how the TNBC cells were affected by CT-transfection through extracellular acidification rate (ECAR)/oxygen consumption rate (OCR) analysis and differential transcription/expression levels. We suggest a novel approach for treating refractory TNBC by an mRNA drug that can exploit metabolic dependencies to exacerbate cell metabolic vulnerability.

Role of pepper MYB transcription factor CaDIM1 in regulation of the drought response.

Abscisic acid (ABA) is a major phytohormone that plays important roles in stress responses, including regulation of gene expression and stomatal closure. Regulation of gene expression by transcription factors is a key cellular process for initiating defense responses to biotic and abiotic stresses. Here, using pepper (Capsicum annuum) leaves, we identified the MYB transcription factor CaDIM1 (Capsicum annuum Drought Induced MYB 1), which was highly induced by ABA and drought stress. CaDIM1 has an MYB domain in the N-terminal region and an acidic domain in the C-terminal region, which are responsible for recognition and transactivation of the target gene, respectively. Compared to control plants, CaDIM1-silenced pepper plants displayed ABA-insensitive and drought-sensitive phenotypes with reduced expression of stress-responsive genes. On the other hand, overexpression of CaDIM1 in Arabidopsis exhibited the opposite phenotypes of CaDIM1-silenced pepper plants, accompanied by enhanced ABA sensitivity and drought tolerance. Taken together, we demonstrate that CaDIM1 functions as a positive regulator of the drought-stress response via modulating ABA-mediated gene expression.

Core Components of Abscisic Acid Signaling and Their Post-translational Modification.

Abscisic acid (ABA) is a major phytohormone that regulates plant growth, development, and abiotic/biotic stress responses. Under stress, ABA is synthesized in various plant organs, and it plays roles in diverse adaptive processes, including seed dormancy, growth inhibition, and leaf senescence, by modulating stomatal closure and gene expression. ABA receptor, clade A protein phosphatase 2C (PP2C), and SNF1-related protein kinase 2 (SnRK2) proteins have been identified as core components of ABA signaling, which is initiated via perception of ABA with receptor and subsequent activation or inactivation by phosphorylation/dephosphorylation. The findings of several recent studies have established that the post-translational modification of these components, including phosphorylation and ubiquitination/deubiquitination, play important roles in regulating their activity and stability. In this review, we discuss the functions of the core components of ABA signaling and the regulation of their activities via post-translational modification under normal and stress conditions.

Pepper Novel Pseudo Response Regulator Protein CaPRR2 Modulates Drought and High Salt Tolerance.

Plants modify their internal states to adapt to environmental stresses. Under environmental stress conditions, plants restrict their growth and development and activate defense responses. Abscisic acid (ABA) is a major phytohormone that plays a crucial role in the osmotic stress response. In osmotic stress adaptation, plants regulate stomatal closure, osmoprotectant production, and gene expression. Here, we isolated CaPRR2 - encoding a pseudo response regulator protein - from the leaves of pepper plants (Capsicum annuum). After exposure to ABA and environmental stresses, such as drought and salt stresses, CaPRR2 expression in pepper leaves was significantly altered. Under drought and salt stress conditions, CaPRR2-silenced pepper plants exhibited enhanced osmotic stress tolerance, characterized by an enhanced ABA-induced stomatal closing and high MDA and proline contents, compared to the control pepper plants. Taken together, our data indicate that CaPRR2 negatively regulates osmotic stress tolerance.

Pepper ubiquitin-specific protease, CaUBP12, positively modulates dehydration resistance by enhancing CaSnRK2.6 stability.

Abscisic acid (ABA) is a plant hormone that activates adaptive mechanisms to environmental stress conditions. Plant adaptive mechanisms are complex and highly modulated processes induced by stress-responsive proteins; however, the precise mechanisms by which these processes function under adverse conditions remain unclear. Here, we isolated CaUBP12 (Capsicum annuum ubiquitin-specific protease 12) from pepper (C. annuum) leaves. We show that CaUBP12 expression is significantly induced after exposure to abiotic stress treatments. We conducted loss-of-function and gain-of-function genetic studies to elucidate the biological functions of CaUBP12 in response to ABA and dehydration stress. CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively; these phenotypes were characterized by regulation of transpirational water loss and stomatal aperture. Under dehydration stress conditions, CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants exhibited lower and higher expression levels of stress-related genes, respectively, than the control plants. We isolated a CaUBP12 interaction protein, CaSnRK2.6, which is a homolog of Arabidopsis OST1; degradation of this protein was partially inhibited by CaUBP12. Similar to CaUBP12-silenced pepper plants and CaUBP12-overexpressing Arabidopsis plants, CaSnRK2.6-silenced pepper plants and CaSnRK2.6-overexpressing Arabidopsis displayed dehydration-sensitive and dehydration-tolerant phenotypes, respectively. Our findings suggest that CaUBP12 positively modulates the dehydration stress response by suppressing CaSnRK2.6 protein degradation.

Pepper Novel Serine-Threonine Kinase CaDIK1 Regulates Drought Tolerance via Modulating ABA Sensitivity.

Adaptation to drought stress is essential for plant growth and development. Plants often adapt to water deficit conditions by activating the ABA signaling. Here, we report that the pepper CaDIK1 (Capsicum annuum Drought Induced Kinase 1) gene is essential for plant tolerance to drought stress. CaDIK1 contains a serine-threonine kinase domain, which plays a role for attachment of phosphate to the target protein. The expression levels of CaDIK1 are upregulated in pepper leaves by ABA, drought, NaCl and H2O2 treatments suggesting its role in abiotic stress response. We used CaDIK1-silenced pepper and CaDIK1-overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to ABA and drought. CaDIK1-silenced pepper plants conferred a reduced ABA sensitivity and drought hypersensitivity, which was accompanied by high levels of transpirational water loss. CaDIK1-OX plants displayed opposite phenotypes to CaDIK1-silenced peppers. In contrast, substitution of Lys350 to Asn in the kinase domain of CaDIK1 did not lead to alteration of drought sensitivity. Collectively, these data indicate that CaDIK1 is a positive regulator of the ABA-mediated drought-stress tolerance.

Functional Analysis of Pepper F-box Protein CaDIF1 and Its Interacting Partner CaDIS1: Modulation of ABA Signaling and Drought Stress Response.

Plant adaptive responses to environmental stress are coordinated by inhibition of plant growth and development. Abscisic acid (ABA) is a major phytohormone that regulates the stress response, and its sensitivity determines stress tolerance levels. In this study, we report the identification and functional role of a novel F-box protein, CaDIF1 (Capsicum annuum Drought-Induced F-box Protein 1). The expression of CaDIF1 in pepper leaves was induced by ABA, drought, H2O2, and NaCl treatments. In comparison with wild-type pepper plants, CaDIF1-silenced pepper plants exhibited a drought-sensitive phenotype, whereas CaDIF1-overexpressing (OX) plants exhibited ABA-sensitive and drought-tolerant phenotypes. Using a yeast two-hybrid assay, we identified CaDIS1 (C. annuum DIF1-Interacting SKP 1), which interacts with CaDIF1 in the cytoplasm and nucleus. Consistent with CaDIF1-silenced pepper plants, CaDIS1-silenced pepper plants displayed ABA insensitivity and reduced drought tolerance, and these were characterized by larger stomatal apertures and greater transpirational water loss. Taken together, our results indicate that CaDIF1 and its interacting partner CaDIS1 synergistically regulate the ABA-dependent defence signaling response to drought stress.

The Pepper Late Embryogenesis Abundant Protein, CaDIL1, Positively Regulates Drought Tolerance and ABA Signaling.

Plants as sessile organisms constantly respond to environmental stress during their growth and development. The regulation of transpiration via stomata plays crucial roles in plant adaptation to drought stress. Many enzyme-encoding genes are involved in regulation of transpiration via modulating stomatal opening and closure. Here, we demonstrate that Capsicum annuum Drought Induced Late embryogenesis abundant protein 1 (CaDIL1) gene is a critical regulator of transpirational water loss in pepper. The expression of CaDIL1 in pepper leaves was upregulated after exposure to abscisic acid (ABA) and drought. Phenotype analysis showed that CaDIL1-silenced pepper and CaDIL1-overexpressing (OX) Arabidopsis transgenic plants exhibited reduced and enhanced drought tolerance, respectively, accompanied by an altered water loss. Furthermore, ABA sensitivity was significantly lower in CaDIL1-silenced pepper, but higher in CaDIL1-OX plants, than that in control plants, which resulted in opposite responses to drought stress in these two plant types. Collectively, our data suggest that CaDIL1 positively regulates the ABA signaling and drought stress tolerance.