GS6, A Member of the GRAS Gene Family,Negatively Regulates Grain Size in Rice

Grain size is an important yield-related trait in rice. Intensive artificial selection for grain size during domestication is evidenced by the larger grains of most of today’s cultivars compared with t...     

CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase,Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.)

Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature (4°C) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.     

Overexpression of Rice Sphingosine-1-Phoshpate Lyase Gene OsSPL1 in Transgenic Tobacco Reduces Salt and Oxidative Stress Tolerance

Sphingolipids, including sphingosine-1-phosphate (S1P), have been shown to function as signaling mediators to regulate diverse aspects of plant growth, development, and stress response. In this study, we performed functional analysis of a rice (Oryza sativa) S1P lyase gene OsSPL1 in transgenic tobacco plants and explored its possible involvement in abiotic stress response. Overexpression of OsSPL1 in transgenic tobacco resulted in enhanced sensitivity to exogenous abscisic acid (ABA), and decreased tolerance to salt and oxidative stress, when compared with the wild type. Furthermore, the expression levels of some selected stress-related genes in OsSPL1-overexpressing plants were reduced after application of salt or oxidative stress, indicating that the altered responsiveness of stress-related genes may be responsible for the reduced tolerance in OsSPL1-overexpressing tobacco plants under salt and oxidative stress. Our results suggest that rice OsSPL1 plays an important role in abiotic stress responses.     

DJ-1 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson''s disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP⁺ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.     

A Putative P-Type ATPase,Apt1, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity of Cryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized the APT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of the APT1 gene resulted in phenotypes consistent with similar roles in C. neoformans. These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and the apt1 mutant strains at either 30°C or 37°C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, and apt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response in C. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningoencephalitis in immunocompromised individuals (44). One million cases of cryptococcosis are estimated to occur each year, and approximately two-thirds of these are fatal (43). Key virulence traits for the fungus include growth at the mammalian host temperature, production of a polysaccharide capsule, deposition of laccase-synthesized melanin in the cell wall, secretion of enzymes, and resistance to host defenses, such as oxidative and nitrosative killing (44).The polysaccharide capsule is a key virulence factor and is both cell associated and released during infection (4). The two species of polysaccharide in the capsule, an abundant glucuronoxylomannan (GXM) and a minor galactoxylomannan (GalXM), cause a number of deleterious effects in mammalian hosts (4, 44). Extracellular vesicles (exosomes) containing capsule polysaccharide are present in culture supernatants, in lysates of macrophages containing C. neoformans, and in association with fungal cells during murine infection (41, 49, 50, 54). These so-called “virulence factor delivery bags” are thought to pass through the cell wall to deliver material outside the cell (50). Proteomic analysis of the vesicles identified 76 proteins, and many of these are associated with virulence, including urease, laccase, heat shock proteins, superoxide dismutase, thiol-specific antioxidants, and catalases (49).The mechanisms of trafficking of capsule polysaccharide and laccase are being actively pursued. For example, analysis of a mutant with a defect in the exocyst GTPase Sec4/Rab8 (designated Sav1) revealed the accumulation of intracellular vesicles containing capsule polysaccharide, thus providing support for intracellular synthesis and secretion via exocytosis (60). In addition, reduced expression of the exocyst protein Sec6 due to RNA interference (RNAi) resulted in partial attenuation of virulence as well as defects in melanin production and the export of urease and soluble capsule polysaccharide (42). The RNAi strains were also completely defective in the production of extracellular exosomes but retained wild-type (WT) levels of cell-associated capsule. Trafficking of the laccase required for melanin production and virulence has also been examined. Hu et al. (25) showed that C. neoformans lacking Vps34 (vacuolar protein sorting 34) had a marked reduction in melanin formation, suggesting that laccase-containing vesicles are derived from the endocytic pathway. Overall, the current evidence suggests that exocytic, endocytic, and specialized extracellular vesicles mediate the export of capsule and other virulence factors in C. neoformans (42, 49, 60).We demonstrated previously that vesicle trafficking functions in C. neoformans are regulated by the cAMP signal transduction pathway, which also controls the elaboration of both the capsule and melanin (28). We found that treatment of C. neoformans with inhibitors of Golgi apparatus-mediated transport (e.g., brefeldin A or monensin) or with lithium chloride results in inhibition of capsule expression (28). In addition, we found that cAMP-dependent protein kinase regulated the expression of a predicted phospatidylethanolamine binding protein, Ova1, which negatively influences capsule and melanin formation. These findings focused our attention on the roles of intracellular trafficking functions and phospholipids in virulence factor expression.In the context of phospholipid trafficking, some aminophospholipid translocases within the P-type ATPases are known to play roles in fungal virulence. For example, the aminophospholipid translocase MgApt2 is required for exocytosis during plant infection by the rice blast pathogen Magnaporthe grisea (18). P-type ATPases are a large family of multitransmembrane domain, ATP-dependent transporters, and three subfamilies are found in eukaryotes (29): (i) heavy metal ion ATPases (e.g., copper transporters), (ii) non-heavy-metal ion ATPases (e.g., Ca²⁺, H⁺, Na⁺, and K⁺ ATPases), and (iii) aminophospholipid translocases (APTs/flippases of the type IV or Drs2 family). APTs maintain the asymmetrical distribution of aminophospholipids in membranes by translocating phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) from one leaflet of the bilayer to the other. Phospholipid asymmetry is important in membrane fusion events (vesicle budding and docking) at the plasma membrane and in the trans-Golgi network (3). Thus, APTs are required for efficient Golgi function and play roles in both endocytosis and exocytosis. Some disorders in humans have been linked or attributed to genes from the APT subfamily, including familial intrahepatic cholestasis and Angelman syndrome (32, 55).Previously, we constructed a deletion of the APT1 gene, encoding a putative aminophospholipid translocase, as part of a study to examine disomy at chromosome 13 in C. neoformans (27). Our preliminary phenotypic analysis suggested a connection to nitrosative stress and prompted further investigation of virulence-related functions. In the present study, we show that Apt1 is functionally related to Drs2 in Saccharomyces cerevisiae and has roles in membrane trafficking and sensitivity to stress (oxidative and nitrosative) and drugs targeting ergosterol biosynthesis and secretion. Importantly, loss of Apt1 does not influence capsule and melanin formation, but the protein is required for intracellular growth in macrophages and for full virulence in mice.     

Cytosolic BolA Plays a Repressive Role in the Tolerance against Excess Iron and MV-Induced Oxidative Stress in Plants

The BolA-like protein is present in all eukaryotes, and it is able to form complex with monothiol glutaredoxin of the same subcellular compartments, suggesting that the BolA-like protein has essential function in eukaryotes, and that the function is associated with its partner glutaredoxin. Some studies have indicated a role for BolA proteins in Fe-S cluster synthesis or in redox homeostasis. However, the physiological function of BolA proteins remains to be elucidated. Here, we report the characterization of an insertion mutant of BolA3 in Arabidopsis. Among the four AtBolA proteins found in Arabidopsis, the AtBolA3 was the only BolA located in the cytosol of plant cells. It was highly expressed in roots. AtBolA3 was able to interact with the cytosolic monothiol glutaredoxin, AtGRXS17. The bola3 mutant did not show any notable phenotype under normal growth condition, but rather grew better than wild type under some stresses. The bola3 mutant was more tolerant to excess iron and the MV-induced oxidative stress than wild type. It displayed no necrosis in leaves, developed longer roots, accumulated more iron and higher Fe-S protein activities in roots. In addition, the mutant possessed a more potent antioxidant defense to scavenge ROS species. Taken together, our data indicated that the cytosolic AtBolA3 has a suppressive role in the tolerance to excess iron and the MV-induced oxidative stress in plants. AtBolA3 seems to be a repressor under some stress conditions.     

Structure and Function of CinD (YtjD) of Lactococcus lactis,a Copper-Induced Nitroreductase Involved in Defense against Oxidative Stress

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k_cat values of 27 and 11 s⁻¹, respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 Å and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.Lactococcus lactis IL1403 is a Gram-positive lactic acid bacterium which is used for the manufacture of food and dairy products but also for an increasing number of biotechnological applications. Given the economical importance of this microorganism, it is often used as a model for molecular studies. Its genome has been sequenced (4), and its proteome has been extensively characterized (11). When applied to industrial processes, this bacterium has to face various stress conditions, such as low pH, high temperature, osmotic shock, and metal stress (44). For instance, in traditional cheese making in Switzerland, L. lactis is exposed to copper released from the copper vats.Copper is an essential micronutrient for both prokaryotes and eukaryotes. The two oxidation states of copper, Cu⁺ and Cu²⁺, allow its participation in many important biological functions. More than 30 enzymes are known to use copper as a cofactor, such as superoxide dismutase (SOD), cytochrome c oxidase, or lysyl oxidase (20). The redox activity of copper can also lead to the generation of free radicals, which cause cellular damage (42, 43). Recently, alternative copper toxicity mechanisms have been demonstrated in bacteria in which copper interferes with the formation of catalytic iron-sulfur clusters (6, 22). Whatever the mechanism of copper toxicity, maintenance of copper homeostasis by controlling the uptake, accumulation, detoxification, and removal of copper is critical for living organisms.Copper homeostasis in L. lactis has not yet been investigated in great detail but appears to resemble the well-characterized copper homeostatic system of Enterococcus hirae (34). L. lactis possesses a copRZA operon, which provides copper resistance. It encodes the CopA copper export ATPase, the CopR copper-inducible repressor, and the CopZ copper chaperone (23). CopR regulates not only the copRZA operon but also an additional 11 genes. This so-called CopR regulon also includes copB, encoding a second putative copper ATPase; lctO, encoding lactate oxidase; and the ydiDE, yahCD-yaiAB, and ytjDBA operons of unknown function. Of all the genes and operons constituting the CopR regulon, the ytjDBA operon was most strongly induced by copper (23). Based on sequence comparison, the first gene of this operon, ytjD, encodes an oxygen-insensitive nitroreductase, which we renamed cinD for copper-induced nitroreductase.Nitroreductases are called oxygen insensitive when they can catalyze the two-electron reduction of nitro compounds in the presence of oxygen. Such enzymes are widespread in nature and are able to reduce a wide range of substrates, such as furazones, nitroaromatic compounds, flavins, and ferricyanide, using NADH or NADPH as the reductant. They are flavoproteins of 22 to 24 kDa and form homodimers with one flavin mononucleotide cofactor per monomer. Although oxygen-insensitive nitroreductases have been extensively studied, their in vivo function remains largely unknown. The closest relative of CinD, which has functionally been studied, is FRP of Vibrio harveyi, with 29% sequence identity to CinD. FRP is not a typical nitroreductase but appears to function as an NADH flavin oxidoreductase which provides reduced flavin to luciferase (19). The next closest relative of CinD, NfsA of Escherichia coli, with 23% sequence identity, exhibits the broad substrate specificity typical of most nitroreductases (48). The structure of this enzyme has been solved at a resolution of 1.7 Å (17). It closely resembles the structures of other enzymes which belong to the oxygen-insensitive nitroreductase family. NfsA has recently been shown to participate in the degradation of 2,4,6-trinitrotoluene (10). This suggests that an important function of nitroreductases could be the metabolism of xenobiotics.We investigated here the structure and function of CinD of L. lactis. CinD was induced by copper, cadmium, and silver and protected L. lactis from oxidative stress exerted by 4-nitroquinoline-N-oxide (NQO). The purified enzyme is a flavoprotein and exhibited nitroreductase activity on NQO and a variety of other substrates, using NADH as the reductant. CinD also possesses catalase activity and is thus able to defend cells against oxidative stress exerted by hydrogen peroxide, xenobiotics, or copper. The three-dimensional structure of CinD was resolved at a 1.35-Å resolution and exhibits a typical nitroreductase structure.     

Sth1p,a Saccharomyces cerevisiae Snf2p/Swi2p homolog,is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins.

The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo. The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity. Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p. The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function. The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways. Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi. These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle.     

Foliar Concentrations of Selected Elements,Assessment of Oxidative Stress Markers and Role of Antioxidant Defense System is Associated with Fly Ash Stress Tolerance in Withania somnifera

Journal of Plant Growth Regulation - Increased dependence on thermal power has resulted in a significant increase in the generation of fly ash (FA), which exacerbates environmental...