Amino acid uptake by temperate tree species characteristic of low- and high-fertility habitats

The relationship between inorganic nitrogen (N) cycling and plant productivity is well established. However, recent research has demonstrated the ability of plants to take up low molecular weight organic N compounds (i.e., amino acids) at rates that often rival those of inorganic N forms. In this study, we hypothesize that temperate forest tree species characteristic of low-fertility habitats will prefer amino acids over species characteristic of high-fertility habitats. We measured the uptake of ¹⁵N-labeled amino acids (glycine, glutamine, arginine, serine), ammonium (NH₄ ⁺), and nitrate (NO₃ ⁻) by four tree species that commonly occur in eastern North America, where their abundances have been correlated with inorganic N availability. Specific uptake rates of amino acids were largely similar for all tree species; however, high-fertility species took up NH₄ ⁺ at rates more than double those of low-fertility species, rendering amino acid N relatively more important to the N nutrition of low-fertility species. Low-fertility species acquired over four times more total N from arginine compared to NH₄ ⁺ and NO₃ ⁻; high-fertility species acquired the most N from NH₄ ⁺. Arginine had the highest uptake rates of any amino acid by all species; there were no significant differences in uptake rates of the remaining amino acids. Our results support the idea that the dominant species in a particular habitat are those best able to utilize the most available N resources.     

Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.     

Functional Characterization of the Rice UDP-glucose 4-epimerase 1, OsUGE1: A Potential Role in Cell Wall Carbohydrate Partitioning during Limiting Nitrogen Conditions

Plants grown under inadequate mineralized nitrogen (N) levels undergo N and carbon (C) metabolic re-programming which leads to significant changes in both soluble and insoluble carbohydrate profiles. However, relatively little information is available on the genetic factors controlling carbohydrate partitioning during adaptation to N-limitation conditions in plants. A gene encoding a uridine-diphospho-(UDP)-glucose 4-epimerase (OsUGE-1) from rice (Oryza sativa) was found to be N-responsive. We developed transgenic rice plants to constitutively over-express the OsUGE-1 gene (OsUGE1-OX1–2). The transgenic rice lines were similar in size to wild-type plants at the vegetative stage and at maturity regardless of the N-level tested. However, OsUGE1-OX lines maintained 18–24% more sucrose and 12–22% less cellulose in shoots compared to wild-type when subjected to sub-optimal N-levels. Interestingly, OsUGE1-OX lines maintained proportionally more galactose and glucose in the hemicellulosic polysaccharide profile of plants compared to wild-type plants when grown under low N. The altered cell wall C-partitioning during N-limitation in the OsUGE1-OX lines appears to be mediated by OsUGE1 via the repression of the cellulose synthesis associated genes, OsSus1, OsCesA4, 7, and 9. This relationship may implicate a novel control point for the deposition of UDP-glucose to the complex polysaccharide profiles of rice cell walls. However, a direct relationship between OsUGE1 and cell wall C-partitioning during N-limitation requires further investigation.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Cytoplasmic alkalinization produced by the combination of anti-immunoglobulin antibody plus cytochalasin D in murine B lymphocytes

The intracellular pH of murine splenic B lymphocytes was measured using the pH-sensitive fluorescent dye, bis(carboxyethyl)carboxyfluorescein (BCECF). After stimulation of B lymphocytes with anti-immunoglobulin antibody plus cytochalasin D, two agents that act in synergy to promote S-phase entry, a late increase in pH was detected that occurred prior to the onset of DNA synthesis. The degree of alkalinization observed was comparable to that produced by two additional mitogenic regimens. Cytoplasmic alkalinization was not blocked by dimethylamiloride. Cytoplasmic alkalinization represents a sign of, and may play a role in, stimulation of B lymphocytes to enter S phase.     

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.