Phenotypic analysis and molecular cloning of discolored-1 (dsc1), a maize gene required for early kernel development

Recessive mutations in the maize dsc1 locus prevent normal kernel development. Solidification of the endosperm in homozygous dsc1– mutant kernels was undetectable 12 days after pollination, at which time the tissue was apparently completely solidified in wild-type kernels. At later times endosperm did solidify in homozygous dsc1– mutant kernels, but there was a marked reduction in the volume of the tissue. Embryo growth in homozygous dsc1– kernels was delayed compared to wild-type kernels, but proceeded to an apparently normal stage 1 in which the scutellum, coleoptile, and shoot apex were clearly defined. Embryo growth then ceased and the embryonic tissues degraded. Late in kernel development no tissue distinctions were obvious in dsc1– mutant embryos. Immature mutant embryos germinated when transplanted from kernels to tissue culture medium prior to embryonic degeneration, but only coleoptile proliferation was observed. The dsc1 gene was isolated by transposon tagging. Analysis of the two different dsc1– mutations confirmed that transposon insertion into the cloned genomic locus was responsible for the observed phenotype. Dsc1 mRNA was detected specifically in kernels 5–7 days after pollination. These data indicate Dsc1 function is required for progression of embryo development beyond a specific stage, and also is required for endosperm development.     

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP₂) levels, consistent with prior evidence that MARCKS can regulate PIP₂ levels. We also found increased staining for PIP₂ in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP₂ co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP₂ levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.     

A maize thiamine auxotroph is defective in shoot meristem maintenance

Plant shoots undergo organogenesis throughout their life cycle via the perpetuation of stem cell pools called shoot apical meristems (SAMs). SAM maintenance requires the coordinated equilibrium between stem cell division and differentiation and is regulated by integrated networks of gene expression, hormonal signaling, and metabolite sensing. Here, we show that the maize (Zea mays) mutant bladekiller1-R (blk1-R) is defective in leaf blade development and meristem maintenance and exhibits a progressive reduction in SAM size that results in premature shoot abortion. Molecular markers for stem cell maintenance and organ initiation reveal that both of these meristematic functions are progressively compromised in blk1-R mutants, especially in the inflorescence and floral meristems. Positional cloning of blk1-R identified a predicted missense mutation in a highly conserved amino acid encoded by thiamine biosynthesis2 (thi2). Consistent with chromosome dosage studies suggesting that blk1-R is a null mutation, biochemical analyses confirm that the wild-type THI2 enzyme copurifies with a thiazole precursor to thiamine, whereas the mutant enzyme does not. Heterologous expression studies confirm that THI2 is targeted to chloroplasts. All blk1-R mutant phenotypes are rescued by exogenous thiamine supplementation, suggesting that blk1-R is a thiamine auxotroph. These results provide insight into the role of metabolic cofactors, such as thiamine, during the proliferation of stem and initial cell populations.     

Altered expression of plant lysyl tRNA synthetase promotes tRNA misacylation and translational recoding of lysine

The Arabidopsis thaliana lysyl tRNA synthetase (AtKRS) structurally and functionally resembles the well-characterized prokaryotic class IIb KRS, including the propensity to aminoacylate tRNA(Lys) with suboptimal identity elements, as well as non-cognate tRNAs. Transient expression of AtKRS in carrot cells promotes aminoacylation of such tRNAs in vivo and translational recoding of lysine at nonsense codons. Stable expression of AtKRS in Zea mays causes translational recoding of lysine into zeins, significantly enriching the lysine content of grain.     

An Approach to Integrating Occupational Safety and Health into Life Cycle Assessment: Development and Application of Work Environment Characterization Factors

Integrating occupational safety and health (OSH) into life cycle assessment (LCA) may provide decision makers with insights and opportunities to prevent burden shifting of human health impacts between the nonwork environment and the work environment. We propose an integration approach that uses industry‐level work environment characterization factors (WE‐CFs) to convert industry activity into damage to human health attributable to the work environment, assessed as disability‐adjusted life years (DALYs). WE‐CFs are ratios of work‐related fatal and nonfatal injuries and illnesses occurring in the U.S. worker population to the amount of physical output from U.S. industries; they represent workplace hazards and exposures and are compatible with the life cycle inventory (LCI) structure common to process‐based LCA. A proof of concept demonstrates application of the WE‐CFs in an LCA of municipal solid waste landfill and incineration systems. Results from the proof of concept indicate that estimates of DALYs attributable to the work environment are comparable in magnitude to DALYs attributable to environmental emissions. Construction and infrastructure‐related work processes contributed the most to the work environment DALYs. A sensitivity analysis revealed that uncertainty in the physical output from industries had the most effect on the WE‐CFs. The results encourage implementation of WE‐CFs in future LCA studies, additional refinement of LCI processes to accurately capture industry outputs, and inclusion of infrastructure‐related processes in LCAs that evaluate OSH impacts.