Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development.

The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the plant meristem and also give rise to all shoot organs. SAM fate is specified by class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, and here we demonstrate that AGO10 specifically interacts with miR166/165. The association is determined by a distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. Notably, the miRNA-binding ability of AGO10, but not its catalytic activity, is required for SAM development, and AGO10 has a higher binding affinity for miR166 than does AGO1, a principal contributor to miRNA-mediated silencing. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP III gene expression.

Multiple ER-to-nucleus stress signaling pathways are activated during Plantago asiatica mosaic virus and Turnip mosaic virus infection in Arabidopsis thaliana.

Pathogens and other adverse environmental conditions can trigger endoplasmic reticulum (ER) stress. ER stress signaling increases the expression of cytoprotective ER-chaperones. The inositol-requiring enzyme (IRE1) is one ER stress sensor that is activated to splice the bZIP60 mRNA that produces a truncated transcription factor that activates gene expression in the nucleus. The IRE1/bZIP60 pathway is associated with restricting potyvirus and potexvirus infection. This study shows that the Plantago asiatica mosaic virus (PlAMV) triple gene block 3 (TGB3) and the Turnip mosaic virus (TuMV) 6K2 proteins activate alternative transcription pathways involving the bZIP17, bZIP28, BAG7, NAC089 and NAC103 factors in Arabidopsis thaliana. Using the corresponding knockout mutant lines, we show that bZIP17, bZIP60, BAG7 and NAC089 are factors in reducing PlAMV infection, whereas bZIP28 and bZIP60 are factors in reducing TuMV infection. We propose a model in which bZIP60 and bZIP17 synergistically induce genes restricting PlAMV infection, while bZIP60 and bZIP28 independently induce genes supporting PlAMV infection. Regarding TuMV-green fluorescent protein (GFP) infection, bZIP60 and bZIP28 serve to repress local and systemic infection. Finally, tauroursodeoxycholic acid treatments were used to demonstrate that the protein folding capacity significantly influences PlAMV accumulation.

Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants.

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.

Trehalose Accumulation Triggers Autophagy during Plant Desiccation.

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis, by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.

Arabidopsis B-cell lymphoma2 (Bcl-2)-associated athanogene 7 (BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29.

To cope with stress and increased accumulation of misfolded proteins, plants and animals use a survival pathway known as the unfolded protein response (UPR) that signals between the endoplasmic reticulum (ER) and the nucleus to maintain cell homeostasis via proper folding of proteins. B-cell lymphoma2 (Bcl-2)-associated athanogene (BAG) proteins are an evolutionarily conserved family of co-chaperones that are linked to disease states in mammals and responses to environmental stimuli (biotic and abiotic) in plants. Molecular and physiological techniques were used to functionally characterize a newly identified branch of the UPR initiated by the ER-localized co-chaperone from Arabidopsis thaliana, AtBAG7. AtBAG7 has functional roles in both the ER and the nucleus. Upon heat stress, AtBAG7 is sumoylated, proteolytically processed and translocated from the ER to the nucleus, where interaction with the WRKY29 transcription factor occurs. Sumoylation and translocation are required for the AtBAG7-WRKY29 interaction and subsequent stress tolerance. In the ER, AtBAG7 interacts with the ER-localized transcription factor, AtbZIP28, and established UPR regulator, the AtBiP2 chaperone. The results indicate that AtBAG7 plays a central regulatory role in the heat-induced UPR pathway.

Proline biosynthesis is required for endoplasmic reticulum stress tolerance in Saccharomyces cerevisiae.

The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP(+)/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.

Cell death control: the interplay of apoptosis and autophagy in the pathogenicity of Sclerotinia sclerotiorum.

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

Programmed cell death occurs asymmetrically during abscission in tomato.

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators.

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment.

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.

AtBAG7, an Arabidopsis Bcl-2-associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response.

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7 was shown to interact directly in vivo with the molecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs.

The unfolded protein response is triggered by a plant viral movement protein.

Infection with Potato virus X (PVX) in Nicotiana benthamiana plants leads to increased transcript levels of several stress-related host genes, including basic-region leucine zipper 60 (bZIP60), SKP1, ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT), and calmodulin (CAM). bZIP60 is a key transcription factor that responds to endoplasmic reticulum (ER) stress and induces the expression of ER-resident chaperones (BiP, PDI, CRT, and CAM). SKP1 is a component of SCF (for SKP1-Cullin-F box protein) ubiquitin ligase complexes that target proteins for proteasomal degradation. Expression of PVX TGBp3 from a heterologous vector induces the same set of genes in N. benthamiana and Arabidopsis (Arabidopsis thaliana) leaves. Virus-induced gene silencing was employed to knock down the expression of bZIP60 and SKP1, and the number of infection foci on inoculated leaves was reduced and systemic PVX accumulation was altered. Silencing bZIP60 led to the suppression of BiP and SKP1 transcript levels, suggesting that bZIP60 might be an upstream signal transducer. Overexpression of TGBp3 led to localized necrosis, but coexpression of TGBp3 with BiP abrogated necrosis, demonstrating that the unfolded protein response alleviates ER stress-related cell death. Steady-state levels of PVX replicase and TGBp2 (which reside in the ER) proteins were unaltered by the presence of TGBp3, suggesting that TGBp3 does not contribute to their turnover. Taken together, PVX TGBp3-induced ER stress leads to up-regulation of bZIP60 and unfolded protein response-related gene expression, which may be important to regulate cellular cytotoxicity that could otherwise lead to cell death if viral proteins reach high levels in the ER.

Bacterial effector HopF2 suppresses arabidopsis innate immunity at the plasma membrane.

Many bacterial pathogens inject a cocktail of effector proteins into host cells through type III secretion systems. These effectors act in concert to modulate host physiology and immune signaling, thereby promoting pathogenicity. In a search for additional Pseudomonas syringae effectors in suppressing plant innate immunity triggered by pathogen or microbe-associated molecular patterns (PAMPs or MAMPs), we identified P. syringae tomato DC3000 effector HopF2 as a potent suppressor of early immune-response gene transcription and mitogen-activated protein kinase (MAPK) signaling activated by multiple MAMPs, including bacterial flagellin, elongation factor Tu, peptidoglycan, lipopolysaccharide and HrpZ1 harpin, and fungal chitin. The conserved surface-exposed residues of HopF2 are essential for its MAMP suppression activity. HopF2 is targeted to the plant plasma membrane through a putative myristoylation site, and the membrane association appears to be required for its MAMP-suppression function. Expression of HopF2 in plants potently diminished the flagellin-induced phosphorylation of BIK1, a plasma membrane-associated cytoplasmic kinase that is rapidly phosphorylated within one minute upon flagellin perception. Thus, HopF2 likely intercepts MAMP signaling at the plasma membrane immediately of signal perception. Consistent with the potent suppression function of multiple MAMP signaling, expression of HopF2 in transgenic plants compromised plant nonhost immunity to bacteria P. syringae pv. Phaseolicola and plant immunity to the necrotrophic fungal pathogen Botrytis cinerea.

Apoptosis-related genes confer resistance to Fusarium wilt in transgenic 'Lady Finger' bananas.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases.

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.

Cytoprotective Co-chaperone BcBAG1 Is a Component for Fungal Development, Virulence, and Unfolded Protein Response (UPR) of Botrytis cinerea.

The Bcl-2 associated athanogene (BAG) family is an evolutionarily conserved group of co-chaperones that confers stress protection against a variety of cellular insults extending from yeasts, plants to humans. Little is known, however, regarding the biological role of BAG proteins in phytopathogenic fungi. Here, we identified the unique BAG gene (BcBAG1) from the necrotrophic fungal pathogen, Botrytis cinerea. BcBAG1 is the homolog of Arabidopsis thaliana AtBAG4, and ectopic expression of BcBAG1 in atbag4 knock-out mutants restores salt tolerance. BcBAG1 deletion mutants (ΔBcbag1) exhibited decreased conidiation, enhanced melanin accumulation and lost the ability to develop sclerotia. Also, BcBAG1 disruption blocked fungal conidial germination and successful penetration, leading to a reduced virulence in host plants. BcBAG1 contains BAG (BD) domain at C-terminus and ubiquitin-like (UBL) domain at N-terminus. Complementation assays indicated that BD can largely restored pathogenicity of ΔBcbag1. Abiotic stress assays showed ΔBcbag1 was more sensitive than the wild-type strain to NaCl, calcofluor white, SDS, tunicamycin, dithiothreitol (DTT), heat and cold stress, suggesting BcBAG1 plays a cytoprotective role during salt stress, cell wall stress, and ER stress. BcBAG1 negatively regulated the expression of BcBIP1, BcIRE1 and the splicing of BcHAC1 mRNA, which are core regulators of unfolded protein response (UPR) during ER stress. Moreover, BcBAG1 interacted with HSP70-type chaperones, BcBIP1 and BcSKS2. In summary, this work demonstrates that BcBAG1 is pleiotropic and not only essential for fungal development, hyphal melanization, and virulence, but also required for response to multiple abiotic stresses and UPR pathway of B. cinerea.

Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development.

Accumulating evidence supports the idea that necrotrophic plant pathogens interact with their hosts by controlling cell death. Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with a broad host range (>400 species). Previously, we established that oxalic acid (OA) is an important pathogenicity determinant of this fungus. In this report, we describe a mechanism by which oxalate contributes to the pathogenic success of this fungus; namely, that OA induces a programmed cell death (PCD) response in plant tissue that is required for disease development. This response exhibits features associated with mammalian apoptosis, including DNA laddering and TUNEL reactive cells. Fungal mutants deficient in OA production are nonpathogenic, and apoptotic-like characteristics are not observed following plant inoculation. The induction of PCD by OA is independent of the pH-reducing abilities of this organic acid, which is required for sclerotial development. Moreover, oxalate also induces increased reactive oxygen species (ROS) levels in the plant, which correlate to PCD. When ROS induction is inhibited, apoptotic-like cell death induced by OA does not occur. Taken together, we show that Sclerotinia spp.-secreted OA is an elicitor of PCD in plants and is responsible for induction of apoptotic-like features in the plant during disease development. This PCD is essential for fungal pathogenicity and involves ROS. Thus, OA appears to function by triggering in the plant pathways responsible for PCD. Further, OA secretion by Sclerotinia spp. is not directly toxic but, more subtly, may function as a signaling molecule.

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress.

The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H(2)O(2), tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Delta(1)-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H(2)O(2) levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.