Translating insights from the seed metabolome into improved prediction for lipid-composition traits in oat (Avena sativa L.)

Oat (Avena sativa L.) seed is a rich resource of beneficial lipids, soluble fiber, protein, and antioxidants, and is considered a healthful food for humans. Little is known regarding the genetic controllers of variation for these compounds in oat seed. We characterized natural variation in the mature seed metabolome using untargeted metabolomics on 367 diverse lines and leveraged this information to improve prediction for seed quality traits. We used a latent factor approach to define unobserved variables that may drive covariance among metabolites. One hundred latent factors were identified, of which 21% were enriched for compounds associated with lipid metabolism. Through a combination of whole-genome regression and association mapping, we show that latent factors that generate covariance for many metabolites tend to have a complex genetic architecture. Nonetheless, we recovered significant associations for 23% of the latent factors. These associations were used to inform a multi-kernel genomic prediction model, which was used to predict seed lipid and protein traits in two independent studies. Predictions for 8 of the 12 traits were significantly improved compared to genomic best linear unbiased prediction when this prediction model was informed using associations from lipid-enriched factors. This study provides new insights into variation in the oat seed metabolome and provides genomic resources for breeders to improve selection for health-promoting seed quality traits. More broadly, we outline an approach to distill high-dimensional “omics” data to a set of biologically meaningful variables and translate inferences on these data into improved breeding decisions.     

Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:     

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors

Applied Microbiology and Biotechnology - The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and...     

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Importin‐α proteins mediate the translocation of nuclear localization signal (NLS)‐containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN‐α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1‐1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense‐regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope‐tagged MOS6 from pathogen‐challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR‐NBS‐type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type‐III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co‐immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN‐α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N‐terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN‐α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.     

The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.     

Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.     

Calcium content of littoral Cladocera in three softwater lakes of the Canadian Shield

Aqueous calcium (Ca) concentrations are declining in softwater lakes of the Canadian Shield largely because of decades of acid deposition and afforestation following timber harvesting. Populations of pelagic cladoceran taxa with high Ca requirements, especially Daphnia spp., are declining in response to reduced aqueous Ca availability. However, the Ca content, and thus the requirements of littoral cladoceran taxa are unknown; therefore, the potential vulnerability of this major component of lake ecosystems to ongoing regional Ca decline remains uncertain. Here, we identify the body Ca concentration of nine littoral cladoceran taxa collected from three lakes in the Muskoka-Haliburton region of Ontario, Canada. Ca content differed among taxonomic groups, ranging from 3.6 mg g⁻¹ for Acroperus harpae to 23.2 mg g⁻¹ for Disparalona spp. Perhaps surprisingly, some littoral microcrustacean taxa have Ca burdens comparable to the Ca-rich daphniids. Therefore, there may be differential responses to ongoing lake water Ca declines among taxa within littoral communities as has been observed among open-water taxa, with potential ecological repercussions for near-shore food webs.     

Large-scale purification,dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK(2)) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V(max) of 1.25 micromol P(i)/min/mg and a K(m) of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.