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In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase. Three soybean genes orthologous to the Arabidopsis lipase gene SUGAR-DEPENDENT1 ( SDP1 ) are upregulated at this time. Suppression of these genes resulted in higher oil levels, with lipid levels in the best lines exceeding 24% of seed weight. In addition, lipase-suppressed lines produced larger seeds compared to wild-type plants, resulting in increases of over 20% in total lipid per seed. Levels of raffinose and stachyose were lower in the transgenic lines, with average reductions of 15% in total raffinose family oligosaccharides observed. Despite the increase in oil, protein content was not negatively impacted and trended higher in the transgenic lines. These results are consistent with a role for SDP1 in turning over TAG to supply carbon for other needs, including the synthesis of oligosaccharides, and offer new strategies to further improve the composition of soybean seeds.

Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some of the proteins that are required for these functions. The proteins and mechanisms for organelle DNA replication are very similar to bacterial or phage systems. The minimal replisome may consist of DNA polymerase, a primase/helicase, and a single-stranded DNA binding protein (SSB), similar to that found in bacteriophage T7. In Arabidopsis, there are two genes for organellar DNA polymerases and multiple potential genes for SSB, but there is only one known primase/helicase protein to date. Genome copy number varies widely between type and age of plant tissues. Replication mechanisms are only poorly understood at present, and may involve multiple processes, including recombination-dependent replication (RDR) in plant mitochondria and perhaps also in chloroplasts. There are still important questions remaining as to how the genomes are maintained in new organelles, and how genome copy number is determined. This review summarizes our current understanding of these processes.

Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.

The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human β-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while Δ mprF2 and Δ mprF1 Δ mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the Δ mprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in Δ mprF2 and Δ mprF1 Δ mprF2 . In the mprF mutants, particularly Δ mprF1 Δ mprF2 , the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the Δ mprF1 Δ mprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both Δ mprF2 and Δ mprF1 Δ mprF2 , compared to the wild type and Δ mprF1 . Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis , including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

Background Plant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana : a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis. Results Yeast-two-hybrid analysis reveals residues 120–295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins. Conclusions The interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function.

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human beta-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild type and mprF mutant strains and show that while delta mprF2 and delta mprF1 delta mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the delta mprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in delta mprF2 and delta mprF1 delta mprF2. In the mprF mutants, particularly delta mprF1 delta mprF2, the decrease in L-PG and PG is compensated by an increase in the phosphorus-containing lipid, GPDGDAG, and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the delta mprF1 delta mprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both delta mprF2 and delta mprF1 delta mprF2, compared to wild type and delta mprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF.Competing Interest StatementThe authors have declared no competing interest.

Granzyme K (GzmK) is a tryptic member of the granzyme family of chymotrypsin-like serine proteases produced by cells of the immune system. Previous studies have indicated that GzmK activates protease-activated receptor 1 (PAR1) enhancing activation of monocytes and wound healing in endothelial cells. Here, we show using peptides and full length proteins that GzmK and, to a lesser extent the related protease GzmA, are capable of activating PAR1 and PAR2. These cleavage events occur at the canonical arginine P1 residue and involve exosite interactions between protease and receptor. Despite cleaving PAR2 at the same point as trypsin, GzmK does not induce a classical Ca 2+ flux but instead activates a distinct signalling cascade, involving recruitment of β-arrestin and phosphorylation of ERK. In epithelial A549 cells, PAR2 activation by GzmK results in the release of inflammatory cytokines IL-6 and IL-8. These data suggest that during an immune response GzmK acts as a pro-inflammatory regulator, rather than as a cytotoxin.

Species with short life spans frequently show a close relationship between population abundance and environmental variation making these organisms potential indicator species of climatic variability. White ( Penaeus setiferus ), brown ( P . aztecus ), and pink ( P . duorarum ) penaeid shrimp typically have an annual life history and are of enormous ecological, cultural, and economic value to the southeastern United States and Gulf of Mexico. Within North Carolina, all three species rely on the Pamlico Sound, a large estuarine system that straddles Cape Hatteras, one of the most significant climate and biogeographic breaks in the world, as a nursery area. These characteristics make penaeid species within the Pamlico Sound a critical species-habitat complex for assessing climate impacts on fisheries. However, a comprehensive analysis of the influence of the environmental conditions that influence penaeid shrimp populations has been lacking in North Carolina. In this study, we used more than 30 years of data from two fishery-independent trawl surveys in the Pamlico Sound to examine the spatial distribution and abundance of adult brown, white, and pink shrimp and the environmental drivers associated with adult shrimp abundance and juvenile brown shrimp recruitment using numerical models. Brown shrimp recruitment models demonstrate that years with higher temperature, salinity, offshore windstress, and North Atlantic Oscillation phase predict increased abundance of juveniles. Additionally, models predicting adult brown, white, and pink shrimp abundance illustrate the importance of winter temperatures, windstress, salinity, the North Atlantic Oscillation index, and the abundance of spawning adult populations from the previous year on shrimp abundance. Our findings show a high degree of variability in shrimp abundance is explained by climate and environmental variation and indicate the importance of understanding these relationships in order to predict the impact of climate variability within ecosystems and develop climate-based adaptive management strategies for marine populations.

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease. Human respiratory bronchioles contain a unique population of secretory cells called respiratory airway secretory cells that are distinct from the cells in the larger proximal airways, and act as unidirectional progenitors for alveolar type 2 cells.

Changing the morphology of a simple epithelial tube to form a highly ramified branching network requires changes in cell behavior that lead to tissue-wide changes in organ shape. How epithelial cells in branched organs modulate their shape and behavior to promote bending and sculpting of the epithelial sheet is not well understood, and the mechanisms underlying this process remain obscure. We show that the Wnt receptor Frizzled 2 (Fzd2) is required for domain branch formation during the initial establishment of the respiratory tree. Live imaging and transcriptome analysis of lung-branching morphogenesis demonstrate that Fzd2 promotes changes in epithelial cell length and shape. These changes in cell morphology deform the developing epithelial tube to generate and maintain new domain branches. Fzd2 controls branch formation and the shape of the epithelial tube by regulating Rho signaling and by the localization of phospho-myosin light chain 2, in turn controlling the changes in the shape of epithelial cells during morphogenesis. This study demonstrates the importance of Wnt/Fzd2 signaling in promoting and maintaining changes in epithelial cell shape that affect development of a branching network.