Multi-omic characterization of the thermal stress phenome in the stony coral Montipora capitata

Background Corals, which form the foundation of biodiverse reef ecosystems, are under threat from warming oceans. Reefs provide essential ecological services, including food, income from tourism, nutrient cycling, waste removal, and the absorption of wave energy to mitigate erosion. Here, we studied the coral thermal stress response using network methods to analyze transcriptomic and polar metabolomic data generated from the Hawaiian rice coral Montipora capitata. Coral nubbins were exposed to ambient or thermal stress conditions over a 5-week period, coinciding with a mass spawning event of this species. The major goal of our study was to expand the inventory of thermal stress-related genes and metabolites present in M. capitata and to study gene-metabolite interactions. These interactions provide the foundation for functional or genetic analysis of key coral genes as well as provide potentially diagnostic markers of pre-bleaching stress. A secondary goal of our study was to analyze the accumulation of sex hormones prior to and during mass spawning to understand how thermal stress may impact reproductive success in M. capitata. Methods M. capitata was exposed to thermal stress during its spawning cycle over the course of 5 weeks, during which time transcriptomic and polar metabolomic data were collected. We analyzed these data streams individually, and then integrated both data sets using MAGI (Metabolite Annotation and Gene Integration) to investigate molecular transitions and biochemical reactions. Results Our results reveal the complexity of the thermal stress phenome in M. capitata, which includes many genes involved in redox regulation, biomineralization, and reproduction. The size and number of modules in the gene co-expression networks expanded from the initial stress response to the onset of bleaching. The later stages involved the suppression of metabolite transport by the coral host, including a variety of sodium-coupled transporters and a putative ammonium transporter, possibly as a response to reduction in algal productivity. The gene-metabolite integration data suggest that thermal treatment results in the activation of animal redox stress pathways involved in quenching molecular oxygen to prevent an overabundance of reactive oxygen species. Lastly, evidence that thermal stress affects reproductive activity was provided by the downregulation of CYP-like genes and the irregular production of sex hormones during the mass spawning cycle. Overall, redox regulation and metabolite transport are key components of the coral animal thermal stress phenome. Mass spawning was highly attenuated under thermal stress, suggesting that global climate change may negatively impact reproductive behavior in this species.

Hornera currieae n. sp. (Cyclostomatida: Horneridae): a new bathyal cyclostome bryozoan with reproductively induced skeletal plasticity

Here we describe a new hornerid, Hornera currieae n. sp. (Bryozoa: Cyclostomatida) from bathyal depths across the New Zealand region. Colonies are irregular, finely branched fans attaining ~40 mm or more in height. Key characters include: (1) thick, semi-hyaline porcellanous skeleton; (2) loss or reduction of nervi (longitudinal striae) away from growing tips; (3) sparse, threadlike cancelli; and (4) small (61–87 µm), widely spaced autozooidal apertures. Diagnostic hornerid traits possessed by H. currieae n. sp. include vertical ancestrular tube, periancestrular budding of daughter zooids, and skeletal ultrastructure dominated by hexagonal semi-nacre grading to pseudofoliated fabric. The abfrontal incubation chamber develops from a cryptic tube arising from the frontally positioned aperture of the fertile zooid. We used SEM, micro-CT and electron backscatter diffractometry (EBSD) to investigate the ultrastructure and internal architecture of H. currieae n. sp. EBSD reveals that crystalline c-axes of laminated crystallites are perpendicular to skeletal walls. Threadlike cancelli, which traverse secondary calcification, connect autozooidal chambers to the colony-wide hypostegal cavity. Micro-CT reveals that abfrontal cancelli usually bend proximally towards the base, but turn distally towards reproductively active regions of the colony in synchrony with gonozooid development. The zone of affected cancelli extends for 4–7 branch internodes below the gonozooid. We assessed whether skeletal ultrastructure was similarly affected, but neither cancellus direction, nor gonozooid proximity, were predictive of the crystallite imbrication direction. We hypothesise that (1) hornerid cancelli are active conduits for colonial metabolite transport and (2) that changes in gradients of metabolites and/or reproductive morphogens within the hypostegal cavity affect cancellus morphogenesis. Potentially, H. currieae n. sp. skeletons may preserve a record of intra-colony metabolite translocation dynamics over time.

Monoacylglycerol disrupts Golgi structure and perilipin 2 association with lipid droplets

The two major products of intestinal triacylglycerol digestion and lipoprotein lipolysis are monoacylglycerols (MAG) and fatty acids. In the gut, these products are taken up by enterocytes and packaged into perilipin-coated cytosolic lipid droplets and then secreted as chylomicrons. We observed that fat feeding or intragastric administration of triacylglycerol oil caused the enterocyte Golgi to fragment into submicron puncta dispersed throughout the cytosol. Further, this apparent Golgi dispersion was also observed in cultured fibroblasts after treatment with fat (cream) and pancreatic lipase, but not when treated with deactivated lipase. We therefore hypothesized that a hydrolytic fat product, specifically monoacylglycerols, fatty acids or a combination of these molecules can trigger Golgi fragmentation. Disruption of coatomer function is known to cause Golgi to fuse with the ER, and blocks perilipin 2 delivery to lipid droplets. Thus, we assessed the effects of MAG on coatomer distribution, Golgi structure and perilipin 2 localization. We found that MAG, but not fatty acids, dispersed coatomer from the Golgi, fragmented the Golgi and caused perilipin 2 to accumulate on cellular membranes. Thus, our findings suggest that monoacylglycerol production during digestion disperses the Golgi, possibly by altering coatomer function, which may regulate metabolite transport between the ER and Golgi.

Engineering cellular metabolite transport for biosynthesis of computationally predicted tropane alkaloid derivatives in yeast

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 μg/L) and 7-fold (172 μg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

Potassium Control of Plant Functions: Ecological and Agricultural Implications

Potassium, mostly as a cation (K+), together with calcium (Ca2+) are the most abundant inorganic chemicals in plant cellular media, but they are rarely discussed. K+ is not a component of molecular or macromolecular plant structures, thus it is more difficult to link it to concrete metabolic pathways than nitrogen or phosphorus. Over the last two decades, many studies have reported on the role of K+ in several physiological functions, including controlling cellular growth and wood formation, xylem–phloem water content and movement, nutrient and metabolite transport, and stress responses. In this paper, we present an overview of contemporary findings associating K+ with various plant functions, emphasizing plant-mediated responses to environmental abiotic and biotic shifts and stresses by controlling transmembrane potentials and water, nutrient, and metabolite transport. These essential roles of K+ account for its high concentrations in the most active plant organs, such as leaves, and are consistent with the increasing number of ecological and agricultural studies that report K+ as a key element in the function and structure of terrestrial ecosystems, crop production, and global food security. We synthesized these roles from an integrated perspective, considering the metabolic and physiological functions of individual plants and their complex roles in terrestrial ecosystem functions and food security within the current context of ongoing global change. Thus, we provide a bridge between studies of K+ at the plant and ecological levels to ultimately claim that K+ should be considered at least at a level similar to N and P in terrestrial ecological studies.

T81. MULTIPLE DRUG USE IN SCHIZOPHRENIA - THE ROLE OF EARLY ENVIRONMENTAL RISK ACCUMULATION AND GENETIC PREDISPOSITION

Background Drug (ab)use and substance use disorders are frequently observed in patients with psychiatric illness, but the underlying causes remain widely unknown. A number of environmental risk factors have been proposed to affect the use of one or multiple drugs in the general population and adolescents. Whereas most previous studies focused on the influence of single risk factors on the use of one or a few selected drugs, the effect of accumulated environmental risk in early life on multiple drug use remains to be studied. Similarly, evidence on genetic susceptibility to the (ab)use of a single drug, e.g. nicotine, alcohol, cocaine, is abundant, while the role of genetic predisposition for multiple drug use - in particular during early life - is yet to be explored. Thus, the current work aims to study the role of environmental as well as genetic risk factors for multiple drug abuse (‘polytoxicomania’) in a large sample of schizophrenic/schizoaffective patients. Methods Information from ~2000 schizophrenia/schizoaffective patients on (preadult) multiple drug use (> 2 drugs) and environmental risk factors was extracted from the Göttingen Research Association for Schizophrenia (GRAS) data collection – currently the largest data base of deeply phenotyped patients with schizophrenia/schizoaffective disorder or other neuropsychiatric diseases. In addition, genetic data from these patients and 2111 healthy blood donors were used in a novel genetic approach that employs multiple genome-wide association studies (GWAS) to identify genetic associations with preadult multiple drug use. Genotyping was performed on a semi-custom Axiom MyDesign Genotyping Array (Affymetrix, Santa Clara, CA, USA), based on a CEU (Caucasian residents of European ancestry from UT, USA) marker backbone. Results The accumulation of environmental risk factors, i.e. sexual abuse, physical abuse, migration, urbanicity, together with alcohol and cannabis consumption as secondary risk factors, in early life (< 18 years) were strongly associated with lifetime multiple drug use (p = 3.48 x 10^-44, extreme group comparison odds ratio (OR) = 31.8). When the sample was split into preadult and adult multiple drug users, there was a remarkable association of the number of preadult environmental risk factors with preadult multiple drug use (p = 1.12 x 10^-25, OR = 243.6). Furthermore, preadult environmental risk accumulation strongly predicted onset of multiple drug use in adulthood (> 18 years; p = 6.27 x 10^-18, OR = 19.4). The application of the novel genetic approach yielded 35 single-nucleotide variants (SNPs) that potentially confer susceptibility to preadult multiple drug use. Out of these, 14 were located in gene-coding regions. Interestingly, 9 of these genes are implicated in neuronal development/function or metabolite transport/transformation. Additional gene-based analyses identified another 4 genes relevant for metabolite transport/transformation as well as 4 genes that play a role in hypoxia signaling. Discussion The present results show that an accumulation of environmental risk factors during early life (< 18 years) is a strong predictor of multiple drug use during adolescence and later life. These findings suggest that exposure to accumulated environmental risk during early life is not only associated with violent aggression – as previously reported by our lab – but is also an important predictor of multiple drug use. Moreover, we present first evidence of a genetic susceptibility to preadult multiple drug use, which will benefit from future replication in suitable samples of patients with mental illness or the general population.

A dynamic anchor domain in slc13 transporters controls metabolite transport

Metabolite transport across cellular membranes is required for bioenergetic processes and metabolic signaling. The solute carrier family 13 (slc13) transporters mediate transport of the metabolites succinate and citrate and hence are of paramount physiological importance. Nevertheless, the mechanisms of slc13 transport and regulation are poorly understood. Here, a dynamic structural slc13 model suggested that an interfacial helix, H4c, which is common to all slc13s, stabilizes the stationary scaffold domain by anchoring it to the membrane, thereby facilitating movement of the SLC13 catalytic domain. Moreover, we found that intracellular determinants interact with the H4c anchor domain to modulate transport. This dual function is achieved by basic residues that alternately face either the membrane phospholipids or the intracellular milieu. This mechanism was supported by several experimental findings obtained using biochemical methods, electrophysiological measurements in Xenopus oocytes, and fluorescent microscopy of mammalian cells. First, a positively charged and highly conserved H4c residue, Arg108, was indispensable and crucial for metabolite transport. Furthermore, neutralization of other H4c basic residues inhibited slc13 transport function, thus mimicking the inhibitory effect of the slc13 inhibitor, slc26a6. Our findings suggest that the positive charge distribution across H4c domain controls slc13 transporter function and is utilized by slc13-interacting proteins in the regulation of metabolite transport.