Arabidopsis Sphingolipid Fatty Acid 2-Hydroxylases (AtFAH1 and AtFAH2) Are Functionally Differentiated in Fatty Acid 2-Hydroxylation and Stress Responses

Abstract

2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b 5 was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-longchain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b 5 more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression.

Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress

“…BI-1 interacts with FAH via the electron donor cytochrome b 5 , which promotes 2-HFA production in Arabidopsis (Nagano et al, , 2012. Moreover, ectopically expressed BI-1 fails to suppress BAXinduced cell death in a S. cerevisiae mutant lacking ScFAH1, suggesting that 2-HFAs are essential for BI-1-mediated cell death suppression.…”

“…Moreover, ectopically expressed BI-1 fails to suppress BAXinduced cell death in a S. cerevisiae mutant lacking ScFAH1, suggesting that 2-HFAs are essential for BI-1-mediated cell death suppression. Our recent study demonstrated that AtFAH1 knockdown plants with a lower content of 2-hydroxy very-long-chain fatty acids (VLCFAs) are hypersensitive to oxidative stress (Nagano et al, 2012). As verified by comprehensive sphingolipidomics by LC-MS/MS (Markham et al, 2006), 2-hydroxy VLCFAs are typical components of plant sphingolipids.…”

“…2-HFAs are typical components of the ceramide backbone of sphingolipids (Imai et al, 1995;Pata et al, 2010). Although many functions of plant sphingolipids remain to be elucidated, accumulating evidence clearly indicates that sphingolipids and their metabolism are closely involved in cell death regulation and various stress responses in plants (Ng et al, 2001;Liang et al, 2003;Townley et al, 2005;Chen et al, 2008Chen et al, , 2012Wang et al, 2008;Saucedo-García et al, 2011;Dutilleul et al, 2012;Kӧnig et al, 2012;Nagano et al, 2012;Mortimer et al, 2013), implying that BI-1 plays a role in cell death regulation through sphingolipid metabolism. Sphingolipids are major components of membrane lipids and are at particularly high concentrations in membrane microdomains, known as lipid rafts in animal cells, which are essential for membranemediated signaling and act as a sorting platform for targeted protein traffic (Simons and Toomre, 2000;Staubach and Hanisch, 2011).…”

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…Both Arabidopsis FAHs (At-FAH1 and At-FAH2) are localized to the endoplasmic reticulum (ER) membrane, and decreasing their levels affects the response to biotic and abiotic stresses (Nagano et al, 2009(Nagano et al, , 2012aKönig et al, 2012). Thus, we hypothesized that FAH could be a useful tool to modify PM microdomains and illuminate the relationship between PM microdomains and innate immunity in plants.…”

“…We have shown that 2-hydroxylation of sphingolipid fatty acids in Arabidopsis thaliana is performed by the enzyme fatty acid 2-hydroxylase (FAH) (Nagano et al, 2009(Nagano et al, , 2012a. Both Arabidopsis FAHs (At-FAH1 and At-FAH2) are localized to the endoplasmic reticulum (ER) membrane, and decreasing their levels affects the response to biotic and abiotic stresses (Nagano et al, 2009(Nagano et al, , 2012aKönig et al, 2012).…”