Macroalgal spore dysfunction: ocean acidification delays and weakens adhesion

Early life stages of marine organisms are predicted to be vulnerable to ocean acidification. For macroalgae, reproduction and population persistence rely on spores to settle, adhere and continue the algal life cycle, yet the effect of ocean acidification on this critical life stage has been largely overlooked. We explicitly tested the biomechanical impact of reduced pH on early spore adhesion. We developed a shear flume to examine the effect of reduced pH on spore attachment time and strength in two intertidal rhodophyte macroalgae, one calcified (Corallina vancouveriensis) and one noncalcified (Polyostea robusta). Reduced pH delayed spore attachment of both species by 40%–52% and weakened attachment strength in C. vancouveriensis, causing spores to dislodge at lower flow‐induced shear forces, but had no effect on the attachment strength of P. robusta. Results are consistent with our prediction that reduced pH disrupts proper curing and gel formation of spore adhesives (anionic polysaccharides and glycoproteins) via protonation and cation displacement, although experimental verification is needed. Our results demonstrate that ocean acidification negatively, and differentially, impacts spore adhesion in two macroalgae. If results hold in field conditions, reduced ocean pH has the potential to impact macroalgal communities via spore dysfunction, regardless of the physiological tolerance of mature thalli.     

Spermatogenesis in Sceloporus variabilis (Squamata,Phrynosomatidae): A non-quiescent pattern

Gaining a deeper understanding of spermatogenic cycles within squamates has aided in our knowledge of the controls of reproduction and has bettered our understanding of reproductive phenology. One of the most studied genera of squamates, Sceloporus, is widely distributed along a latitudinal and elevational gradient in temperate, tropical, low-elevation and high-elevation habitats. Due to this wide distribution and varying habitats, Sceloporus exhibit differences in their spermatogenic activity (including both cyclical and acyclical patterns) and may be one of the most useful genera for understanding the abiotic correlations with spermatogenesis. The spermatogenic activity in Sceloporus variabilis was studied histologically (in a population that inhabits a tropical region at Los Tuxtlas, Veracruz, Mexico) and found to exhibit a unique cyclical pattern with an extended period of maximum activity (from November to July) and the absence of regression and quiescence. Furthermore, these data corroborate previous works on the spermatogenic cycles of S. variabilis despite different populations utilised. These data suggest that although abiotic factors may play a role in the timing of spermatogenesis, phylogenetic signal may be equally as important. More data concerning spermatogenic cycles in phylogenetically related taxa from differing habitats will elucidate the patterns of spermatogenic diversity.     

Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

An examination of targeted gene neighborhoods in strawberry

Background  

Strawberry (Fragaria spp.) is the familiar name of a group of economically important crop plants and wild relatives that also represent an emerging system for the study of gene and genome evolution. Its small stature, rapid seed-to-seed cycle, transformability and miniscule basic genome make strawberry an attractive system to study processes related to plant physiology, development and crop production; yet it lacks substantial genomics-level resources. This report addresses this deficiency by characterizing 0.71 Mbp of gene space from a diploid species (F. vesca). The twenty large genomic tracks (30-52 kb) captured as fosmid inserts comprise gene regions with roles in flowering, disease resistance, and metabolism.     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.