ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activities

Kimberlin, Athen; Han, Gongshe; Luttgeharm, Kyle D.; Chen, Ming; Cahoon, Rebecca E.; Stone, Julie M.; Markham, Jonathan E.; Dunn, Teresa M.; Cahoon, Edgar B.

doi:10.1104/pp.16.00965

Cited by 33 publications

(69 citation statements)

References 54 publications

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“…We favor the first mechanism, in which ORM binds to the SPT complex with the ssSPT interaction, based on a recent study by Kimberlin et al (2016). In addition, we observed that the ORMs and ssSPT localize in the ER, which could be the site where they interact (Figure 1; Supplemental Figure 6).…”

Section: Discussionmentioning

confidence: 70%

Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis

Yin

Rong

et al. 2016

The Plant Cell

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Serine palmitoyltransferase (SPT), a pyridoxyl-59-phosphate-dependent enzyme, catalyzes the first and rate-limiting step in sphingolipid biosynthesis. In humans and yeast, orosomucoid proteins (ORMs) negatively regulate SPT and thus play an important role in maintaining sphingolipid levels. Despite the importance of sphingoid intermediates as bioactive molecules, the regulation of sphingolipid biosynthesis through SPT is not well understood in plants. Here, we identified and characterized the Arabidopsis thaliana ORMs, ORM1 and ORM2. Loss of function of both ORM1 and ORM2 (orm1 amiR-ORM2) stimulated de novo sphingolipid biosynthesis, leading to strong sphingolipid accumulation, especially of long-chain bases and ceramides. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays confirmed that ORM1 and ORM2 physically interact with the small subunit of SPT (ssSPT), indicating that ORMs inhibit ssSPT function. We found that orm1 amiR-ORM2 plants exhibited an early-senescence phenotype accompanied by H 2 O 2 production at the cell wall and in mitochondria, active vesicular trafficking, and formation of cell wall appositions. Strikingly, the orm1 amiR-ORM2 plants showed increased expression of genes related to endoplasmic reticulum stress and defenses and also had enhanced resistance to oxidative stress and pathogen infection. Taken together, our findings indicate that ORMs interact with SPT to regulate sphingolipid homeostasis and play a pivotal role in environmental stress tolerance in plants.

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Section: Discussionmentioning

confidence: 70%

Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis

Yin

Rong

et al. 2016

The Plant Cell

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“…One of the interesting observations made prior to this analysis was the differential activity in the two classes of ceramide synthase resulting from a perturbation to the level of ORMs in the system. Previous studies postulated that this differential activity potentially points to a possible regulatory interaction between ORMs and the ceramide synthases [19].…”

Section: The Effect Of Perturbations On the Generated Modelmentioning

confidence: 97%

“…To predict plausible regulatory schemes capable of reproducing all of the observed responses, we started with a set of 23 schemes that had been postulated in previous works [19,22].…”

Section: Predicting the Regulatory Scheme Of The Sphingolipid Pathwaymentioning

confidence: 99%

“…In addition, previous work on the role of ORMs on the sphingolipid pathway in plants had hypothesized the possibility of a regulatory interaction between ORMs and ceramide synthases [19]. Therefore, the set of starting regulatory networks to be tested included different combinations…”

Section: Predicting the Regulatory Scheme Of The Sphingolipid Pathwaymentioning

confidence: 99%

“…ORMs were proposed to act as modulators of ceramide synthases [19]. It was shown that the overexpression of ORM genes leads to differential activity between the two classes of ceramide synthase.…”

Section: Introductionmentioning

confidence: 99%

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Dissecting the regulatory roles of ORM proteins in the sphingolipid pathway of plants

Alsiyabi

González-Solís

Cahoon

et al. 2020

Preprint

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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 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.

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Disruption of long‐chain base hydroxylation alters growth and impacts sphingolipid synthesis in Physcomitrella patens

et al. 2021

Self Cite

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Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella 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 sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content in sbh protonemata 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. The sbh lines 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 of sbh lines 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 bryophyte P. patens and angiosperm A. thaliana.

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ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activities

Cited by 33 publications

References 54 publications

Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis

Orosomucoid Proteins Interact with the Small Subunit of Serine Palmitoyltransferase and Contribute to Sphingolipid Homeostasis and Stress Responses in Arabidopsis

Dissecting the regulatory roles of ORM proteins in the sphingolipid pathway of plants

Disruption of long‐chain base hydroxylation alters growth and impacts sphingolipid synthesis in Physcomitrella patens

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