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The serine palmitoyltransferase (SPT) complex catalyzes the first and rate-limiting step in sphingolipid biosynthesis in all eukaryotes. ORM/ORMDL proteins are negative regulators of SPT that respond to cellular sphingolipid levels. However, the molecular basis underlying ORM/ORMDL-dependent homeostatic regulation of SPT is not well understood. We determined the cryo–electron microscopy structure ofArabidopsisSPT-ORM1 complex, composed of LCB1, LCB2a, SPTssa, and ORM1, in an inhibited state. A ceramide molecule is sandwiched between ORM1 and LCB2a in the cytosolic membrane leaflet. Ceramide binding is critical for the ORM1-dependent SPT repression, and dihydroceramides and phytoceramides differentially affect this repression. A hybrid β sheet, formed by the amino termini of ORM1 and LCB2a and induced by ceramide binding, stabilizes the amino terminus of ORM1 in an inhibitory conformation. Our findings provide mechanistic insights into sphingolipid homeostatic regulation via the binding of ceramide to the SPT-ORM/ORMDL complex that may have implications for plant-specific processes such as the hypersensitive response for microbial pathogen resistance.

 

The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of −5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom. 

Systems-level analyses, such as differential gene expression analysis, co-expression analysis, and metabolic pathway reconstruction, depend on the accuracy of the transcriptome. Multiple tools exist to perform transcriptome assembly from RNAseq data. However, assembling high quality transcriptomes is still not a trivial problem. This is especially the case for non-model organisms where adequate reference genomes are often not available. Different methods produce different transcriptome models and there is no easy way to determine which are more accurate. Furthermore, having alternative-splicing events exacerbates such difficult assembly problems. While benchmarking transcriptome assemblies is critical, this is also not trivial due to the general lack of true reference transcriptomes.

In this study, we first provide a pipeline to generate a set of the simulated benchmark transcriptome and corresponding RNAseq data. Using the simulated benchmarking datasets, we compared the performance of various transcriptome assembly approaches including both de novo and genome-guided methods. The results showed that the assembly performance deteriorates significantly when alternative transcripts (isoforms) exist or for genome-guided methods when the reference is not available from the same genome. To improve the transcriptome assembly performance, leveraging the overlapping predictions between different assemblies, we present a new consensus-based ensemble transcriptome assembly approach, ConSemble.

Without using a reference genome, ConSemble using four de novo assemblers achieved an accuracy up to twice as high as any de novo assemblers we compared. When a reference genome is available, ConSemble using four genome-guided assemblies removed many incorrectly assembled contigs with minimal impact on correctly assembled contigs, achieving higher precision and accuracy than individual genome-guided methods. Furthermore, ConSemble using de novo assemblers matched or exceeded the best performing genome-guided assemblers even when the transcriptomes included isoforms. We thus demonstrated that the ConSemble consensus strategy both for de novo and genome-guided assemblers can improve transcriptome assembly. The RNAseq simulation pipeline, the benchmark transcriptome datasets, and the script to perform the ConSemble assembly are all freely available from:http://bioinfolab.unl.edu/emlab/consemble/.

 

Sphingolipids are a vital component of plant cellular endomembranes and carry out multiple functional and regulatory roles. Different sphingolipid species confer rigidity to the membrane structure, facilitate trafficking of secretory proteins, and initiate programmed cell death. Although the regulation of the sphingolipid pathway is yet to be uncovered, increasing evidence has pointed to orosomucoid proteins (ORMs) playing a major regulatory role and potentially interacting with a number of components in the pathway, including both enzymes and sphingolipids. However, experimental exploration of new regulatory interactions is time consuming and often infeasible. In this work, a computational approach was taken to address this challenge. A metabolic network of the sphingolipid pathway in plants was reconstructed. The steady-state rates of reactions in the network were then determined through measurements of growth and cellular composition of the different sphingolipids in Arabidopsis seedlings. The Ensemble modeling framework was modified to accurately account for activation mechanisms and subsequently used to generate sets of kinetic parameters that converge to the measured steady-state fluxes in a thermodynamically consistent manner. In addition, the framework was appended with an additional module to automate screening the parameters and to output models consistent with previously reported network responses to different perturbations. By analyzing the network’s response in the presence of different combinations of regulatory mechanisms, the model captured the experimentally observed repressive effect of ORMs on serine palmitoyltransferase (SPT). Furthermore, predictions point to a second regulatory role of ORM proteins, namely as an activator of class II (or LOH1 and LOH3) ceramide synthases. This activating role was found to be modulated by the concentration of free ceramides, where an accumulation of these sphingolipid species dampened the activating effect of ORMs on ceramide synthase. The predictions pave the way for future guided experiments and have implications in engineering crops with higher biotic stress tolerance. 

Virtually all land plants are coated in a cuticle, a waxy polyester that prevents nonstomatal water loss and is important for heat and drought tolerance. Here, we describe a likely genetic basis for a divergence in cuticular wax chemistry between Sorghum bicolor , a drought tolerant crop widely cultivated in hot climates, and its close relative Zea mays (maize). Combining chemical analyses, heterologous expression, and comparative genomics, we reveal that: 1) sorghum and maize leaf waxes are similar at the juvenile stage but, after the juvenile-to-adult transition, sorghum leaf waxes are rich in triterpenoids that are absent from maize; 2) biosynthesis of the majority of sorghum leaf triterpenoids is mediated by a gene that maize and sorghum both inherited from a common ancestor but that is only functionally maintained in sorghum; and 3) sorghum leaf triterpenoids accumulate in a spatial pattern that was previously shown to strengthen the cuticle and decrease water loss at high temperatures. These findings uncover the possibility for resurrection of a cuticular triterpenoid-synthesizing gene in maize that could create a more heat-tolerant water barrier on the plant’s leaf surfaces. They also provide a fundamental understanding of sorghum leaf waxes that will inform efforts to divert surface carbon to intracellular storage for bioenergy and bioproduct innovations. 

Abstract Lipid structures affect membrane biophysical properties such as thickness, stability, permeability, curvature, fluidity, asymmetry, and interdigitation, contributing to membrane function. Sphingolipids are abundant in plant endomembranes and plasma membranes (PMs) and comprise four classes: ceramides, hydroxyceramides, glucosylceramides, and glycosylinositolphosphoceramides (GIPCs). They constitute an array of chemical structures whose distribution in plant membranes is unknown. With the aim of describing the hydrophobic portion of sphingolipids, 18 preparations from microsomal (MIC), vacuolar (VM), PM, and detergent-resistant membranes (DRM) were isolated from Arabidopsis (Arabidopsis thaliana) leaves. Sphingolipid species, encompassing pairing of long-chain bases and fatty acids, were identified and quantified in these membranes. Sphingolipid concentrations were compared using univariate and multivariate analysis to assess sphingolipid diversity, abundance, and predominance across membranes. The four sphingolipid classes were present at different levels in each membrane: VM was enriched in glucosylceramides, hydroxyceramides, and GIPCs; PM in GIPCs, in agreement with their key role in signal recognition and sensing; and DRM in GIPCs, as reported by their function in nanodomain formation. While a total of 84 sphingolipid species was identified in MIC, VM, PM, and DRM, only 34 were selectively distributed in the four membrane types. Conversely, every membrane contained a different number of predominant species (11 in VM, 6 in PM, and 17 in DRM). This study reveals that MIC, VM, PM, and DRM contain the same set of sphingolipid species but every membrane source contains its own specific assortment based on the proportion of sphingolipid classes and on the predominance of individual species. 

Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. InPhyscomitrella patens, 4‐hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long‐chain base (LCB). To assess the functional significance of t18:0, CRISPR‐Cas9 mutagenesis was used to generate mutant lines lacking the soleSPHINGOID BASE HYDROXYLASE(SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content insbhprotonemata was 2.4‐fold higher than in wild‐type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl‐chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl‐chain pairing in glucosylceramides, in which 4,8‐sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. Thesbhlines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content ofsbhlines decreased to wild‐type levels, and the mutants exhibited phenotypes more similar to wild‐type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyteP. patensand angiospermA. thaliana.

 

Plant fatty acids are used for food, feed, fuel, and industrial materials. Structurally and chemically diverse fatty acids, referred to as unusual or specialized fatty acids, are found in the seed oils of diverse plant species. Many unusual fatty acids have potential use as alternative and renewable sources of biofuels and bio‐based industrial feedstocks due to their variant structures' physical or functional properties. Oils enriched in these fatty acids can increase the value of oilseed crops and provide co‐products that can be readily extracted from lignocellulosic materials in biomass crops. Here, we describe recent progress in strategies for enhancement of oil production and quality in oilseed crops and the extension of this knowledge for metabolic engineering of biomass crops. Successful implementation of these strategies, in part, through the use of emerging synthetic biology tools, will provide added product value for the economic sustainability of biomass crops as bioenergy feedstocks.