The elastic modulus of Matrigel as determined by atomic force microscopy

Recent studies indicate that the biophysical properties of the cellular microenvironment strongly influence a variety of fundamental cell behaviors. The extracellular matrix’s (ECM) response to mechanical force, described mathematically as the elastic modulus, is believed to play a particularly critical role in regulatory and pathological cell behaviors. The basement membrane (BM) is a specialization of the ECM that serves as the immediate interface for many cell types (e.g. all epithelial cells) and through which cells are connected to the underlying stroma. Matrigel is a commercially available BM-like complex and serves as an easily accessible experimental simulant of native BMs. However, the local elastic modulus of Matrigel has not been defined under physiological conditions. Here we present the procedures and results of indentation tests performed on Matrigel with atomic force microscopy (AFM) in an aqueous, temperature controlled environment. The average modulus value was found to be approximately 450 Pa. However, this result is considerably higher than macroscopic shear storage moduli reported in the scientific literature. The reason for this discrepancy is believed to result from differences in test methods and the tendency of Matrigel to soften at temperatures below 37° C.     

Transfer RNA of a collagen producing tissue,chick-embryo calvaria

Transfer RNa was isolated from calvaria prepared from chick embryos incubated for 15–17 days. The chargeability of the unfractionated tRNA with ten amino acids tested was very similar to that of unfractionated tRNA from adult chicken liver when data were expressed on the basis of pmoles of amino acceptance per A₂₆₀ unit of tRNA. However, the relative amount of tRNA in calvaria is only about one-fourth the amount in liver. Analysis of individual species of tRNA by two-dimensional electrophoresis on polyacrylamide gels shows that there are fewer isoaccepting species of tRNA in calvaria than in liver.     

Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines

Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) and close relatives produce a variety of structurally diverse volatile and non‐volatile specialized (‘secondary’) metabolites, including terpenes, flavonoids and acyl sugars. A genetic screen is described here to profile leaf trichome and surface metabolite extracts of nearly isogenic chromosomal substitution lines covering the tomato genome. These lines contain specific regions of the Solanum pennellii LA0716 genome in an otherwise ‘wild‐type’ M82 tomato genetic background. Regions that have an impact on the total amount of extractable mono‐ and sesquiterpenes (IL2‐2) or only sesquiterpenes (IL10‐3) or specifically influence accumulation of the monoterpene α‐thujene (IL1‐3 and IL1‐4) were identified using GC‐MS. A rapid LC‐TOF‐MS method was developed and used to identify changes in non‐volatile metabolites through non‐targeted analysis. Metabolite profiles generated using this approach led to the discovery of introgression lines producing different acyl chain substitutions on acyl sugar metabolites (IL1‐3/1‐4 and IL8‐1/8‐1‐1), as well as two regions that influence the quantity of acyl sugars (IL5‐3 and IL11‐3). Chromosomal region 1‐1/1‐1‐3 was found to influence the types of glycoalkaloids that are detected in leaf surface extracts. These results show that direct chemical screening is a powerful way to characterize genetic diversity in trichome specialized metabolism.     

Striking Natural Diversity in Glandular Trichome Acylsugar Composition Is Shaped by Variation at the Acyltransferase2 Locus in the Wild Tomato Solanum habrochaites

Acylsugars are polyesters of short- to medium-length acyl chains on sucrose or glucose backbones that are produced in secretory glandular trichomes of many solanaceous plants, including cultivated tomato (Solanum lycopersicum). Despite their roles in biotic stress adaptation and their wide taxonomic distribution, there is relatively little information about the diversity of these compounds and the genes responsible for their biosynthesis. In this study, acylsugar diversity was assessed for 80 accessions of the wild tomato species Solanum habrochaites from throughout the Andes Mountains. Trichome metabolites were analyzed by liquid chromatography-time of flight-mass spectrometry, revealing the presence of at least 34 structurally diverse acylsucroses and two acylglucoses. Distinct phenotypic classes were discovered that varied based on the presence of glucose or sucrose, the numbers and lengths of acyl chains, and the relative total amounts of acylsugars. The presence or absence of an acetyl chain on the acylsucrose hexose ring caused clustering of the accessions into two main groups. Analysis of the Acyltransferase2 gene (the apparent ortholog of Solyc01g105580) revealed differences in enzyme activity and gene expression correlated with polymorphism in S. habrochaites accessions that varied in acylsucrose acetylation. These results are consistent with the hypothesis that glandular trichome acylsugar acetylation is under selective pressure in some populations of S. habrochaites and that the gene mutates to inactivity in the absence of selection.Trichomes are specialized epidermal cells that protrude from the surface of a variety of plant tissues. They are thought to protect against environmental stresses such as herbivory (Kang et al., 2010a; Weinhold and Baldwin, 2011), loss of water through transpiration, and UV irradiation (Zhou et al., 2007). In particular, secreting glandular trichomes (SGTs) serve as “chemical factories” where specialized metabolites are produced, stored, or volatized (Wagner, 1991; Schilmiller et al., 2008, 2010a). In addition, SGTs produce and secrete proteins on the plant surface for insect protection (Yu et al., 1992; Thipyapong et al., 1997) and pathogen defense (Shepherd et al., 2005). SGTs also contribute to the taste and smell of plants by releasing volatile metabolites. For example, the distinctive aromas of many Mediterranean herbs of the Lamiaceae (mint family) derive from SGTs (Schilmiller et al., 2008), and compounds from the glands of hops (Humulus lupulus in the Cannabaceae) contribute to beer flavor and aroma (Wang et al., 2008). Furthermore, a number of SGT-borne metabolites are commercially valuable, especially for pharmaceutical purposes. For example, artemisinin, a widely used antimalarial, is a sesquiterpene lactone from the trichomes of Artemisia annua (Liu et al., 2011). In addition to their value in foods and medicines, trichomes provide excellent models for analyzing biosynthetic enzymes and pathways (Schilmiller et al., 2008, 2009, 2012b; Bohlmann and Gershenzon, 2009; Sallaud et al., 2009).Plants in the genus Solanum include important crop species such as potato (Solanum tuberosum), eggplant (Solanum melongena), and tomato (Solanum lycopersicum). Previous studies reported that SGTs of cultivated tomato and its wild relatives accumulate high levels of exudates containing a variety of specialized metabolites, for example flavonoids, alkaloids, and terpenoids (Wagner, 1991; Schilmiller et al., 2008, 2010a; McDowell et al., 2011). Cultivated tomato and its wild relatives have morphologically and chemically diverse trichomes. For example, Luckwill (1943) defined seven morphologically distinguishable types of trichomes in plants of this genus, including four glandular types (types 1, 4, 6, and 7; Supplemental Fig. S1; for more Solanum spp. trichome images, see Kang et al., 2010a, 2010b). The presence of specific types of trichomes and their densities vary across species and even within a single plant according to tissue types, developmental stages, and environmental conditions (Werker, 2000; Li et al., 2004). These morphologically distinct SGTs vary in the amounts and types of metabolites that they produce, accumulate, and/or secrete (Werker, 2000). For example, S. lycopersicum M82 leaf type 6 SGTs accumulate the sesquiterpenes β-caryophyllene and α-humulene, while the glands on the stem lack these metabolites (Schilmiller et al., 2010b). There are also species- and accession-specific differences in SGT metabolite profiles. For instance, methylketones accumulate in type 6 glands of a subset of Solanum habrochaites accessions (Fridman et al., 2005; Yu et al., 2010). Similarly, acylglucoses are highly abundant in type 4 glands of Solanum pennellii LA0716, while acylsucroses predominate in S. lycopersicum and S. habrochaites (Shapiro et al., 1994; McDowell et al., 2011). The chemical and morphological diversity of trichomes in different Solanum species and accessions makes the genus an attractive target for the identification of diverse trichome-borne metabolites and the major biosynthetic pathways responsible for their synthesis operating in each trichome type.The value of the comparative metabolomics approach in trichomes was recently demonstrated in studies of Solanum spp. trichome monoterpene and sesquiterpene biosynthesis (Bohlmann and Gershenzon, 2009; Sallaud et al., 2009; Schilmiller et al., 2009). It was discovered that S. lycopersicum SGTs synthesize monoterpenes from the cis-prenyldiphosphate intermediate neryldiphosphate (Sallaud et al., 2009; Schilmiller et al., 2009). This is contrary to the previous paradigm, where the trans-prenyldiphosphate geranyldiphosphate was considered the universal intermediate for monoterpene biosynthesis. An analogous example of biosynthetic innovation was reported for SGTs of S. habrochaites LA1777 (Sallaud et al., 2009), shown to produce sesquiterpenes in the plastid using the all-cis-prenyldiphosphate substrate Z,Z-farnesyldiphosphate. This is counter to the commonly described cytosolic sesquiterpene pathway, which uses the all-trans-sesquiterpene synthase substrate E,E-farnesyldiphosphate. Furthermore, a recent study demonstrated chemical diversity of trichome terpenes in geographically distinct S. habrochaites accessions associated with the evolution of terpene synthases, revealing how the plasticity of biosynthetic enzymes contributes to chemical complexity and diversity (Gonzales-Vigil et al., 2012). These observations suggest that trichome specialized metabolism is evolutionarily plastic, perhaps due to selective pressure from insects or other environmental stress agents.Acylsugars are sticky exudates made in SGTs that are thought to physically or chemically improve plant defense (Mirnezhad et al., 2010; Weinhold and Baldwin, 2011). Results from the literature indicate strong acylsugar diversity in various Solanum spp. trichomes (Schilmiller et al., 2010a, 2010b; McDowell et al., 2011). Acylsugars are categorized as either Suc or Glc esters based on the type of sugar core (Fig. 1), and they also have varying numbers and lengths of acyl chains decorating the sugar moiety. In particular, S. pennellii accumulates enormous amounts of acylsugars, up to 20% of leaf dry weight (Fobes et al., 1985). In addition, previously published data showed that total acylsugars in geographically distinct S. pennellii accessions vary in quantity, the proportion of Suc or Glc backbones, and the overall types of fatty acid esters (FAs) on the sugars (Shapiro et al., 1994). However, this study did not identify specific acylsugar types.Open in a separate windowFigure 1.Structural classes of acylsugars in Solanum species. A, Schematic structure of an acylglucose. The structure shown depicts a Glc triester composed of Glc and three acyl chains with various numbers of carbons represented as R. B, Schematic structure of an acylsucrose. The proposed structure shows a Suc tetraester with three acyl chains on the Glc ring and one on the Fru ring. If the sugar moiety is decorated with three, four, or five acyl chains, it is referred to as a Suc triester, tetraester, or pentaester, respectively. The positions of the acyl chains are currently unknown, with the exception of the most abundant acylsugar in cultivated tomato (M82) that was structurally characterized by NMR (Schilmiller et al., 2010a). In addition, a few acylsugars were isolated and reported from S. habrochaites and other species by King et al. (1990, 1993). Note the changes in nomenclature since these papers were published: Lycopersicum typicum LA1777 is now called S. habrochaites LA1777, and Lycopersicum hirsutum has been changed to S. habrochaites.To explore the detailed acylsugar chemotypes within accessions of one species, we focused on 80 accessions collected throughout the geographical range of S. habrochaites in Peru and Ecuador (Supplemental Table S1). We describe differences in sugar backbone as well as numbers and lengths of acyl chains, including the presence or absence of an acetyl group, which we found to be a major difference in accessions from the southern and northern Andes Mountains. The recent identification of the acyltransferase2 enzyme (SlAT2; encoded by Solyc01g105580), involved in acylsucrose biosynthesis in S. lycopersicum (Schilmiller et al., 2012a), permitted a test of the hypothesis that differences in expression or activity of this enzyme play an important role in the chemical diversity observed. The results extend previous evidence that Solanum spp. SGT chemistry is highly dynamic (Gonzales-Vigil et al., 2012) and show that the AT2 gene is surprisingly diverse across populations of S. habrochaites.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

Saline systems of the Great Plains of western Canada: an overview of the limnogeology and paleolimnology

In much of the northern Great Plains, saline and hypersaline lacustrine brines are the only surface waters present. As a group, the lakes of this region are unique: there is no other area in the world that can match the concentration and diversity of saline lake environments exhibited in the prairie region of Canada and northern United States. The immense number of individual salt lakes and saline wetlands in this region of North America is staggering. Estimates vary from about one million to greater than 10 million, with densities in some areas being as high as 120 lakes/km². Despite over a century of scientific investigation of these salt lakes, we have only in the last twenty years advanced far enough to appreciate the wide spectrum of lake types, water chemistries, and limnological processes that are operating in the modern settings. Hydrochemical data are available for about 800 of the lake brines in the region. Composition, textural, and geochemical information on the modern bottom sediments has been collected for just over 150 of these lakes. Characterization of the biological and ecological features of these lakes is based on even fewer investigations, and the stratigraphic records of only twenty basins have been examined. The lake waters show a considerable range in ionic composition and concentration. Early investigators, concentrating on the most saline brines, emphasized a strong predominance of Na⁺ and SO₄ ^-2 in the lakes. It is now realized, however, that not only is there a complete spectrum of salinities from less than 1 ppt TDS to nearly 400 ppt, but also virtually every water chemistry type is represented in lakes of the region. With such a vast array of compositions, it is difficult to generalize. Nonetheless, the paucity of Cl-rich lakes makes the northern Great Plains basins somewhat unusual compared with salt lakes in many other areas of the world (e.g., Australia, western United States). Compilations of the lake water chemistries show distinct spatial trends and regional variations controlled by groundwater input, climate, and geomorphology. Short-term temporal variations in the brine composition, which can have significant effects on the composition of the modern sediments, have also been well documented in several individual basins. From a sedimentological and mineralogical perspective, the wide range of water chemistries exhibited by the lakes leads to an unusually large diversity of modern sediment composition. Over 40 species of endogenic precipitates and authigenic minerals have been identified in the lacustrine sediments. The most common non-detrital components of the modern sediments include: calcium and calcium-magnesium carbonates (magnesian calcite, aragonite, dolomite), and sodium, magnesium, and sodium-magnesium sulfates (mirabilite, thenardite, bloedite, epsomite). Many of the basins whose brines have very high Mg/Ca ratios also have hydromagnesite, magnesite, and nesquehonite. Unlike salt lakes in many other areas of the world, halite, gypsum, and calcite are relatively rare endogenic precipitates in the Great Plains lakes. The detrital fraction of the lacustrine sediments is normally dominated by clay minerals, carbonate minerals, quartz, and feldspars. Sediment accumulation in these salt lakes is controlled and modified by a wide variety of physical, chemical, and biological processes. Although the details of these modern sedimentary processes can be exceedingly complex and difficult to discuss in isolation, in broad terms, the processes operating in the salt lakes of the Great Plains are ultimately controlled by three basic factors or conditions of the basin: (a) basin morphology; (b) basin hydrology; and (c) water salinity and composition. Combinations of these parameters interact to control nearly all aspects of modern sedimentation in these salt lakes and give rise to four 'end member' types of modern saline lacustrine settings in the Great Plains: (a) clastics-dominated playas; (b) salt-dominated playas; (c) deep water, non-stratified lakes; and (d) deep water, "permanently" stratified lakes.