The Essential Nature of Sphingolipids in Plants as Revealed by the Functional Identification and Characterization of theArabidopsisLCB1 Subunit of Serine Palmitoyltransferase

Abstract

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

“…The lcb1 mutations caused a substantially reduced level of the LCB2 protein in both a Chinese hamster ovary cell mutant line [43] and yeast cells [44], thus leading to lethality in both cases. Similarly, the lcb1-1 mutation causes an embryolethal phenotype, indicating an essential role of FBR11 in plant growth and development [41]. However, a striking phenotype of fbr11-1 appears to be its resistance to FB 1 .…”

“…Sequence comparison revealed that FBR11 encodes a putative LCB1 subunit of SPT, catalyzing the formation of 3-ketosphinganine during sphingolipid de novo synthesis (Figure 4). During the course of this study, Chen et al [41] reported the characterization of LCB1/FBR11. Coexpression of LCB1/FBR11 and LCB2 cDNA was able to complement the long-chain base auxotrophy of S. cerevisiae lcb1∆ and lcb2∆ single and double mutants, indicating that both LCB1 and LCB2 are functional subunits of SPT.…”

“…Coexpression of LCB1/FBR11 and LCB2 cDNA was able to complement the long-chain base auxotrophy of S. cerevisiae lcb1∆ and lcb2∆ single and double mutants, indicating that both LCB1 and LCB2 are functional subunits of SPT. Moreover, lcb1-1, a stronger mutant allele than fbr11-1, was suggested to cause abnormal embryo development [41].…”

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activities

“…SPT synthesizes LCBs, unique components of sphingolipids, by catalyzing a pyridoxal phosphate-dependent condensation of Ser and palmitoyl (16:0)-CoA in plants (Markham et al, 2013). Similar to other eukaryotes, the Arabidopsis (Arabidopsis thaliana) SPT is a heterodimer consisting of LCB1 and LCB2 subunits (Chen et al, 2006;Dietrich et al, 2008;Teng et al, 2008). Research to date has shown that SPT is regulated primarily by posttranslational mechanisms involving physical interactions with noncatalytic, membrane-associated proteins that confer positive and negative regulation of SPT activity (Han et al, 2009Breslow et al, 2010).…”

“…In plants, the maintenance of sphingolipid homeostasis is vital to ensure sufficient sphingolipids for growth (Chen et al, 2006;Kimberlin et al, 2013) while restricting the accumulation of PCD-inducing ceramides and LCBs until required for processes such as the pathogen-triggered hypersensitive response. Serine palmitoyltransferase (SPT), which catalyzes the first step in LCB synthesis, is generally believed to be the primary control point for sphingolipid homeostasis (Hanada, 2003).…”