Jeremy L. Dahmen

The long and short of it: temperature-dependent modifications of fatty acid chain length and unsaturation in the galactolipid profiles of the diatoms Haslea ostrearia and Phaeodactylum tricornutum

The purpose of this study was to examine the effect of different growth temperatures on the fatty acid compositions of the photosynthetically important galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), of the “blue” pennate diatom, Haslea ostrearia, and the model pennate diatom, Phaeodactylum tricornutum, with the hypothesis that their sn-2 fatty acids would be modulated in the same manner as for dinoflagellates. Positive-ion electrospray ionization/mass spectrometry/mass spectrometry was used to characterize the galactolipids of each diatom. At 20°C, H. ostrearia and P. tricornutum were rich in eicosapentaenoic acid (EPA; C20:5) at the sn-1 position and in C16 fatty acids at the sn-2 position of MGDG and DGDG. At 30°C, however, H. ostrearia and P. tricornutum contained no EPA or other C20 fatty acids, but rather contained higher percentages of C18 fatty acids at sn-1. At 30°C, no galactolipid in either diatom contained more than three unsaturations on any of its fatty acids. While these two species differ in galactolipid composition, they both possess a similar method of acclimating their galactolipids to a higher growth temperature: reducing the numbers of the longest and shortest fatty acid chains, as well as decreasing the total number of unsaturations.

Predicting gene regulatory networks of soybean nodulation from RNA-Seq transcriptome data

BackgroundHigh-throughput RNA sequencing (RNA-Seq) is a revolutionary technique to study the transcriptome of a cell under various conditions at a systems level. Despite the wide application of RNA-Seq techniques to generate experimental data in the last few years, few computational methods are available to analyze this huge amount of transcription data. The computational methods for constructing gene regulatory networks from RNA-Seq expression data of hundreds or even thousands of genes are particularly lacking and urgently needed.ResultsWe developed an automated bioinformatics method to predict gene regulatory networks from the quantitative expression values of differentially expressed genes based on RNA-Seq transcriptome data of a cell in different stages and conditions, integrating transcriptional, genomic and gene function data. We applied the method to the RNA-Seq transcriptome data generated for soybean root hair cells in three different development stages of nodulation after rhizobium infection. The method predicted a soybean nodulation-related gene regulatory network consisting of 10 regulatory modules common for all three stages, and 24, 49 and 70 modules separately for the first, second and third stage, each containing both a group of co-expressed genes and several transcription factors collaboratively controlling their expression under different conditions. 8 of 10 common regulatory modules were validated by at least two kinds of validations, such as independent DNA binding motif analysis, gene function enrichment test, and previous experimental data in the literature.ConclusionsWe developed a computational method to reliably reconstruct gene regulatory networks from RNA-Seq transcriptome data. The method can generate valuable hypotheses for interpreting biological data and designing biological experiments such as ChIP-Seq, RNA interference, and yeast two hybrid experiments.

Mono- and digalactosyldiacylglycerol composition of the marennine-producing diatom, Haslea ostrearia: Comparison to a selection of pennate and centric diatoms

Diatoms are one of the largest groups of primary producers in the oceans, yet despite their environmental importance little is known about their plastidial lipid biochemistry. It has been previously reported that Skeletonema species contain primarily C16/C16 and C20/C16 forms of mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Likewise, it was also reported that Phaeodactylum tricornutum contains primarily C16/C16 and C20/C20 forms of MGDG and DGDG. We seek to relate their studies to other diatoms, both in the centrics and pennates, with particular focus on the marennine‐producing pennate diatom, Haslea ostrearia. To this end, the composition and positional distribution of fatty acids of MGDG and DGDG were examined using positive‐ion electrospray ionization/mass spectrometry (ESI/MS). Two centric diatoms, Skeletonema marinoi and Thalassiosira weissflogii, and the pennate diatom, P. tricornutum, contained primarily C20/C16 (sn‐1/sn‐2) and C18/C16 forms of MGDG and DGDG. The other pennate diatoms, H. ostrearia and Navicula perminuta, contained primarily C18/C16 or C18/C18 forms of MGDG and DGDG, indicating a previously unrecognized fatty acid diversity in diatom MGDG and DGDG.

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. VI. Biochemical and genomic comparison of galactolipid biosynthesis between Chromera velia (Chromerida), a photosynthetic alveolate with red algal plastid ancestry, and the dinoflagellate, Lingulodinium polyedrum

Chromera velia is a recently discovered, photosynthetic, free-living alveolate that is the closest free-living relative to non-photosynthetic apicomplexan parasites. Most plastids, regardless of their origin, have membranes composed chiefly of two galactolipids, mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Because of the hypothesized shared red algal origin between the plastids of C. velia and dinoflagellates, our primary objectives were to examine how growth temperature affects MGDG and DGDG composition via positive-ion electrospray/mass spectrometry (ESI/MS) and positive ion/electrospray/mass spectrometry/mass spectrometry (ESI/MS/MS), and to examine galactolipid biosynthetic genes to determine if shared ancestry translates into shared MGDG and DGDG composition. When growing at 20°C, C. velia produces eicosapentaenoic acid-rich 20:5(n-3)/20:5(n-3) (sn-1/sn-2) MGDG and 20:5(n-3)/20:5(n-3) DGDG as its primary galactolipids, with relative percentage compositions of approximately 35 and 60%, respectively. At 30°C these are lessened by approximately 5 and 8%, respectively, by the corresponding production of 20:5/20:4 forms of these lipids. The presence of 20:5 at the sn-1 position is similar to what has been observed previously in a cluster of peridinin-containing dinoflagellates, but the presence of 20:5(n-3) at the sn-2 position is extremely rare. Thus, the forms of MGDG and DGDG in C. velia displayed similarities and differences to what has been observed in peridinin-containing dinoflagellates, such as Lingulodinium polyedrum, which produces 20:5/18:5 and 20:5/18:4 as the major forms of MGDG and DGDG. We develop conceptual models from the galactolipids observed and galactolipid-relevant gene annotations to explain the presence of polyunsaturated fatty acid-containing MGDG and DGDG in both L. polyedrum and C. velia.

Interfacing whispering gallery mode optical microresonator biosensors with the plant defense elicitor chitin.

The biomaterial class of chitooligosaccharides (chitin), commonly found in insects and fungi, is one of the most abundant on earth. Substantial evidence implicates chitin in mediating a diverse array of plant cellular signaling events, including the induction of plant defense mechanisms against invading pests. However, these recognition and mediation mechanisms, including the binding kinetics between chitin and their plant recognition receptors, are not fully understood. Therefore, the creation of a platform capable of both interfacing with chitin and plant cell receptors, and monitoring their interactions, would significantly advance our understanding of this plant defense elicitor. Recently, a label-free, highly sensitive biosensor platform, based on Whispering Gallery Mode optical microresonators, has been developed to study such biomolecular interactions. Here, we demonstrate how this unique platform can be interfaced with chitin using simple carbohydrate chemistry. The surface chemistry is demonstrated using X-ray photoelectron spectroscopy, fluorescence microscopy, optical profilometry, ellipsometry, and contact angle measurements. The resulting surface is uniform, with an average surface roughness of 1.25nm, and is active toward chitin recognition elements. Optical loss measurements using standard quantitative cavity analysis techniques demonstrate that the bioconjugated platforms maintain the high performance (Q>10(6)) required to track binding interactions in this system. The platform is able to detect lectin, which binds COs, at 10μg/mL concentration. This biosensor platforms unique capabilities for label-free, high sensitivity biodetection, when properly interfaced with the biomaterials of interest, could provide the basis for a robust analytical technique to probe the binding dynamics of chitin-plant cell receptors.

Squishy lipids: Temperature effects on the betaine and galactolipid profiles of a C18/C18 peridinin‐containing dinoflagellate, Symbiodinium microadriaticum (Dinophyceae), isolated from the mangrove jellyfish, Cassiopea xamachana

Previous work from our laboratory has shown dinoflagellates, which possess the carotenoid peridinin, have been divided into two clusters based on plastid galactolipid fatty acid composition. In one cluster major forms of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), lipids that comprise the majority of photosynthetic membranes, were C18/C18 (sn‐1/sn‐2), with octadecapentaenoic [18:5(n‐3)] and octadecatetraenoic [18:4(n‐3)] acid as principal fatty acids. The other cluster contained C20/C18 major forms, with eicosapentaenoic acid [20:5(n‐3)] being the predominant sn‐1 fatty acid. In this study, we have found that Symbiodinium microadriaticum isolated from the jellyfish, Cassiopea xamachana, when grown at 30°C, produced MGDG and DGDG with a more saturated fatty acid, 18:4(n‐3), at the sn‐2 carbon than when grown at 20°C where 18:5(n‐3) predominates. This modulation of the sn‐2 fatty acids level of saturation is mechanistically similar to what has been observed in Pyrocystis, a C20/C18 dinoflagellate. We have also examined the effect of growth temperature on the betaine lipid, diacylglycerylcarboxyhydroxymethylcholine (DGCC), which has been observed by others to be the predominant non plastidial polar lipid in dinoflagellates. Temperature effects on it were minimal, with very few modulations in fatty acid unsaturation as observed in MGDG and DGDG. Rather, the primary difference seen at the two growth temperatures was the alteration of the amount of minor forms of DGCC, as well as a second betaine lipid, diacylglyceryl‐N,N,N‐trimethylhomoserine.

A Soybean Acyl Carrier Protein, GmACP, Is Important for Root Nodule Symbiosis

Legumes (members of family Fabaceae) establish a symbiotic relationship with nitrogen-fixing soil bacteria (rhizobia) to overcome nitrogen source limitation. Single root hair epidermal cells serve as the entry point for bacteria to infect the host root, leading to development of a new organ, the nodule, which the bacteria colonize. In the present study, the putative role of a soybean acyl carrier protein (ACP), GmACP (Glyma18g47950), was examined in nodulation. ACP represent an essential cofactor protein in fatty acid biosynthesis. Phylogenetic analysis of plant ACP protein sequences showed that GmACP was classified in a legume-specific clade. Quantitative reverse-transcription polymerase chain reaction analysis demonstrated that GmACP was expressed in all soybean tissues but showed higher transcript accumulation in nodule tissue. RNA interference-mediated gene silencing of GmACP resulted in a significant reduction in nodule numbers on soybean transgenic roots. Fluorescent protein-labeled GmACP was localized to plastids in planta, the site of de novo fatty acid biosynthesis in plants. Analysis of the fatty acid content of root tissue silenced for GmACP expression, as determined by gas chromatography-mass spectrometry, showed an approximately 22% reduction, specifically in palmitic and stearic acid. Taken together, our data provide evidence that GmACP plays an important role in nodulation.

Interfacing whispering gallery mode microresonators for environmental biosensing

Label-free biosensors that combine high sensitivity and high specificity characteristics have shown tremendous potential for applications in medical diagnostics, and have more recently been extended to the food safety and environmental monitoring arenas. A unique type of label-free, optical biosensor, based on Whispering Gallery Mode microresonators, has tremendous potential to revolutionize biodetection due to its extreme sensitivity. The primary limitation of these biosensors, however, is that they require the addition of biorecognition elements to specifically target a biological species of interest. Therefore, the ability to selectively functionalize the microresonator for a specific target molecule, without degrading device performance, is extremely important, and represents the next step in translating these devices from laboratory to field environments. Here, we demonstrate a variety of straightforward bioconjugation strategies that not only impart specificity to optical microresonators, but also allow for the creation of multi-use platforms for complex environments. Of particular interest is the ability to detect harmful bacteria, insects, and fungi in crop and water systems. The resulting surface chemistries are illustrated with XPS, SEM, and fluorescence and optical microscopy, and the device sensitivity is determined via quantitative microcavity analysis. The ability to minimize non-specific adsorption and target unique molecules in complex environments is demonstrated via ellipsometry and in situ device testing. The resulting devices can be recycled several times without loss of sensitivity. By combining these high sensitivity biosensors with appropriate biochemistries, the resulting platforms can be extended to address broader issues in environmental biosensing that directly impact agriculture.

Mono- and digalactosyldiacylglycerol composition of dinoflagellates. VII. Evidence against galactolipid production and plastid presence in the heterotrophic, basal dinoflagellate, Oxyrrhis marina

The heterotrophic genus Oxyrrhis contains two species, O. marina and O. maritima, which occupy positions basal to the dinoflagellate lineage. Oxyrrhis is also related to apicomplexan parasites, which have recently been shown to have a non-photosynthetic, relic plastid referred to as the apicoplast. A recent study by Slamovits & Keeling (2008) demonstrated the presence of plastid-targeted proteins within O. marina. We hypothesized that if O. marina does indeed have plastids, then mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), which are the two most prominent plastidial membrane lipids, would be present. Therefore, we examined three isolates of O. marina to determine if they can produce MGDG and DGDG. We observed that O. marina, when fed the chlorophyte Dunaliella tertiolecta, possessed forms of MGDG and DGDG containing a C18:3 fatty acid at the sn-1 position and most containing a C16:3-4 fatty acid at the sn-2 position; these were derived solely from the prey itself. Examination of published expressed sequence tag (EST) and transcriptome databases of O. marina for the genes encoding MGDG and DGDG synthases, two enzymes integral to the incorporation of galactose in the final forms of these lipids, failed to reveal their presence. Taken together, these results indicate that O. marina does not produce a non-photosynthetic, relictual plastid. However, the presence of plastid-targeted proteins may indicate that O. marina maintains, however briefly, plastids acquired from its prey in a form of kleptoplasty that has been observed previously in dinoflagellates.

Current and emerging analytical technologies for analyzing chitin-protein binding interactions

Abstract Chitin, a small organic molecule commonly found in fungal cell walls and insect exoskeletons, has the ability to elicit an immune response in plants by binding to specific membrane-bound receptors. Understanding how plants detect and fend off deleterious fungi and insects will enable improved defense strategies against these pathogens. A wide array of techniques, including affinity binding studies, isothermal calorimetry, structural analysis, and molecular genomic methods have been used to identify and characterize chitin-binding receptors as well as the kinetic parameters of chitin-receptor interactions. There are a number of newly developed analytical technologies in mechanical, electrochemical, and optical biosensing that have great potential to further elucidate the interactions between chitin and its binding partners. In this review, we provide a detailed examination of the methods currently used to characterize chitin-binding interactions, along with emerging analytical techniques that have the potential to transform this area of study.