The DNA polymerase λ is required for the repair of non-compatible DNA double strand breaks by NHEJ in mammalian cells

DNA polymerase lambda (polλ) is a recently identified DNA polymerase whose cellular function remains elusive. Here we show, that polλ participates at the molecular level in a chromosomal context, in the repair of DNA double strand breaks (DSB) via non-homologous end joining (NHEJ) in mammalian cells. The expression of a catalytically inactive form of polλ (polλDN) decreases the frequency of NHEJ events in response to I-Sce-I-induced DSB whereas inactivated forms of its homologues polβ and polμ do not. Only events requiring DNA end processing before ligation are affected; this defect is associated with large deletions arising in the vicinity of the induced DSB. Furthermore, polλDN-expressing cells exhibit increased sensitization and genomic instability in response to ionizing radiation similar to that of NHEJ-defective cells. Our data support a requirement for polλ in repairing a subset of DSB in genomic DNA, thereby contributing to the maintenance of genetic stability mediated by the NHEJ pathway

Involvement of DNA polymerase μ in the repair of a specific subset of DNA double-strand breaks in mammalian cells

The repair of DNA double-strand breaks (DSB) requires processing of the broken ends to complete the ligation process. Recently, it has been shown that DNA polymerase μ (polμ) and DNA polymerase λ (polλ) are both involved in such processing during non-homologous end joining in vitro. However, no phenotype was observed in animal models defective for either polμ and/or polλ. Such observations could result from a functional redundancy shared by the X family of DNA polymerases. To avoid such redundancy and to clarify the role of polμ in the end joining process, we generated cells over-expressing the wild type as well as an inactive form of polμ (polμD). We observed that cell sensitivity to ionizing radiation (IR) was increased when either polμ or polμD was over-expressed. However, the genetic instability in response to IR increased only in cells expressing polμD. Moreover, analysis of intrachromosomal repair of the I-SceI-induced DNA DSB, did not reveal any effect of either polμ or polμD expression on the efficiency of ligation of both cohesive and partially complementary ends. Finally, the sequences of the repaired ends were specifically affected when polμ or polμD was over-expressed, supporting the hypothesis that polμ could be involved in the repair of a DSB subset when resolution of junctions requires some gap filling

Nitrogen Management in Grasslands and Forage-Based Production Systems–Role of Biological Nitrification Inhibition (BNI)

Nitrogen (N), being the most critical and essential nutrient for plant growth, largely determines the productivity in both extensive- and intensive- grassland systems. Nitrification and denitrification processes in the soil are the primary drivers generating reactive-N: NO3-, N2O, and NO, and is largely responsible for N-loss and degradation of grasslands. Suppressing nitrification can thus facilitate the retention of soil-N to sustain long-term productivity of grasslands and forage-based production systems. Certain plants can suppress soil nitrification by releasing inhibitors from roots, a phenomenon termed ‘biological nitrification inhibition’ (BNI). Recent methodological developments (e.g. bioluminescence assay to detect BNIs from plant-root systems) led to significant advances in our ability to quantify and characterize BNI function in pasture grasses. Among grass-pastures, BNI-capacity is strongest in low-N adapted grasses such as Brachiaria humidicola and weakest in high-N environment grasses such as Italian ryegrass (Lolium perenne) and B. brizantha. The chemical identity of some of the BNIs produced in plant tissues and released from roots has now been established and their mode of inhibitory action determined on nitrifying bacteria Nitrosomonas. Synthesis and release of BNIs is a highly regulated and localized process, triggered by the presence of NH4+ in the rhizosphere, which facilitates the release of BNIs close to soil-nitrifier sites. Substantial genotypic variation is found for BNI-capacity in B. humidicola, which opens the way for its geneticmanipulation. Field studies suggest that Brachiaria grasses suppress nitrification and N2O emissions from soil. The potential for exploiting BNI function (from a genetic improvement and a system perspective) to develop production systems that are low-nitrifying, low N2O-emitting, economically efficient and ecologically sustainable, will be the subject of discussion

The ongoing search for the molecular basis of plant osmosensing

Introduction: Cell viability and metabolism depend on cytoplasmic water and solute content, and organisms have evolved mechanisms to sense changes in cell water content, solute concentrations, cell volume, and/or turgor. This Perspective addresses the response to osmotic challenge in land plants and describes their special dependence on cellular water status for growth and development. Understanding how plants cope with water limitation may allow us to mitigate the agricultural effects of drought, a critical limitation on global crop productivity that is likely to increase in severity as the climate changes (Long and Ort, 2010). The signaling pathways by which plants respond to osmotic challenge are intriguing from an evolutionary standpoint: some aspects of these pathways resemble those of fungal or mammalian cells, some are similar to prokaryotic mechanisms, and yet others are unique to plants (as described below and in Hamann, 2012). In addition to the importance of osmotic homeostasis in land plants, we will discuss some of the specific context and language of plant stress biology, and describe what is known (and not known) about the molecular pathways by which plants sense and respond to osmotic challenges

Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses

Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components

Kinetics of error generation in homologous B-family DNA polymerases

The kinetics of forming a proper Watson–Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all ∼2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase

Evidence that abscisic acid promotes degradation of SNF1-related protein kinase (SnRK) 1 in wheat and activation of a putative calcium-dependent SnRK2

Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) form a major family of signalling proteins in plants and have been associated with metabolic regulation and stress responses. They comprise three subfamilies: SnRK1, SnRK2, and SnRK3. SnRK1 plays a major role in the regulation of carbon metabolism and energy status, while SnRKs 2 and 3 have been implicated in stress and abscisic acid (ABA)-mediated signalling pathways. The burgeoning and divergence of this family of protein kinases in plants may have occurred to enable cross-talk between metabolic and stress signalling, and ABA-response-element-binding proteins (AREBPs), a family of transcription factors, have been shown to be substrates for members of all three subfamilies. In this study, levels of SnRK1 protein were shown to decline dramatically in wheat roots in response to ABA treatment, although the amount of phosphorylated (active) SnRK1 remained constant. Multiple SnRK2-type protein kinases were detectable in the root extracts and showed differential responses to ABA treatment. They included a 42 kDa protein that appeared to reduce in response to 3 h of ABA treatment but to recover after longer treatment. There was a clear increase in phosphorylation of this SnRK2 in response to the ABA treatment. Fractions containing this 42 kDa SnRK2 were shown to phosphorylate synthetic peptides with amino acid sequences based on those of conserved phosphorylation sites in AREBPs. The activity increased 8-fold with the addition of calcium chloride, indicating that it is calcium-dependent. The activity assigned to the 42 kDa SnRK2 also phosphorylated a heterologously expressed wheat AREBP

A nucleotide binding rectification Brownian ratchet model for translocation of Y-family DNA polymerases

Y-family DNA polymerases are characterized by low-fidelity synthesis on undamaged DNA and ability to catalyze translesion synthesis over the damaged DNA. Their translocation along the DNA template is an important event during processive DNA synthesis. In this work we present a Brownian ratchet model for this translocation, where the directed translocation is rectified by the nucleotide binding to the polymerase. Using the model, different features of the available structures for Dpo4, Dbh and polymerase ι in binary and ternary forms can be easily explained. Other dynamic properties of the Y-family polymerases such as the fast translocation event upon dNTP binding for Dpo4 and the considerable variations of the processivity among the polymerases can also be well explained by using the model. In addition, some predicted results of the DNA synthesis rate versus the external force acting on Dpo4 and Dbh polymerases are presented. Moreover, we compare the effect of the external force on the DNA synthesis rate of the Y-family polymerase with that of the replicative DNA polymerase

Overexpression of a Common Wheat Gene TaSnRK2.8 Enhances Tolerance to Drought, Salt and Low Temperature in Arabidopsis

Drought, salinity and low temperatures are major factors limiting crop productivity and quality. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) plays a key role in abiotic stress signaling in plants. In this study, TaSnRK2.8, a SnRK2 member in wheat, was cloned and its functions under multi-stress conditions were characterized. Subcellular localization showed the presence of TaSnRK2.8 in the cell membrane, cytoplasm and nucleus. Expression pattern analyses in wheat revealed that TaSnRK2.8 was involved in response to PEG, NaCl and cold stresses, and possibly participates in ABA-dependent signal transduction pathways. To investigate its role under various environmental stresses, TaSnRK2.8 was transferred to Arabidopsis under control of the CaMV-35S promoter. Overexpression of TaSnRK2.8 resulted in enhanced tolerance to drought, salt and cold stresses, further confirmed by longer primary roots and various physiological characteristics, including higher relative water content, strengthened cell membrane stability, significantly lower osmotic potential, more chlorophyll content, and enhanced PSII activity. Meanwhile, TaSnRK2.8 plants had significantly lower total soluble sugar levels under normal growing conditions, suggesting that TaSnRK2.8 might be involved in carbohydrate metabolism. Moreover, the transcript levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI3, ABI4, ABI5), stress-responsive genes, including two ABA-dependent genes (RD20A, RD29B) and three ABA-independent genes (CBF1, CBF2, CBF3), were generally higher in TaSnRK2.8 plants than in WT/GFP controls under normal/stress conditions. Our results suggest that TaSnRK2.8 may act as a regulatory factor involved in a multiple stress response pathways