Cloning and molecular characterisation of a Δ8-sphingolipid-desaturase from Nicotiana tabacum closely related to Δ6-acyl-desaturases

Abstract

Investigation on the absence of Delta(6)-desaturase activity in Nicotiana tabacum has led to the cloning of a new desaturase gene from this organism (NTDXDES) that exhibited unexpected biochemical activity. Cladistic analysis shows clustering of NTDXDES together with functional Delta(6)-acyl-desaturases of near Solanales plants, such as Borago and Echium. This group lies apart from that of previously characterised Delta(8)-sphingolipid-desaturases, which also includes two putative tobacco members identified in this study. Moreover, strong expression of NTDXDES is found in leaves, flowers, fruits and developing seeds of tobacco plants that is highly dependent on the development phase, with transcriptional activity being higher at stages of active tissue growth. This pattern is similar to that showed by Delta(6)-acyl-desaturases characterised in Boraginaceae species. However, functional assays using a yeast expression system revealed that the protein encoded by NTDXDES lacks Delta(6)-desaturase activity, but instead it is able to desaturate sphingolipid substrates by introducing a double bond on the Delta(8)-position. These data indicate that NTDXDES represent a novel desaturase gene placed in a different evolutionary lineage to that of previously characterised Delta(8)-desaturases.

Rapid nanoscale quantitative analysis of plant sphingolipid long-chain bases by GC-MS

“…Although slight, but significant, dissimilarities in the LCB profiles of plant leaves exist between our data and those of others [17,21,33], they are unlikely due to technical issues but rather reflect differences in the physiological status of plants between studies. Indeed, the fact that several samples allow for a good match between our results and the literature supports such a hypothesis.…”

Isolation and Functional Characterisation of the Genes Encoding Δ8-Sphingolipid Desaturase from Brassica rapa

“…Previously, studies on plant D 8 -sphingolipid desaturase genes such as in B. officinalis and in N. tabacum were focused on only one copy of the gene. Results of these studies showed that the D 8 -sphingolipid desaturase genes were primarily expressed in active developmental tissues (Sperling et al, 2001;García-Maroto et al, 2007). Another study showed that RnD8A and RnD8B were expressed in all the tissues of R. nigrum, but the expression patterns of these two genes were investigated together because they showed very high sequence identities (Song et al, 2010).…”

“…Genes encoding plant D 8 -sphingolipid desaturases have been identified from Arabidopsis thaliana, Brassica napus, Helianthus annuus, Borago officinalis, Aquilegia vulgaris, Nicotiana tabacum, Stylosanthes hamata and Ribes nigrum (Sperling et al, 1998(Sperling et al, , 2000(Sperling et al, , 2001Michaelson et al, 2002;García-Maroto et al, 2007;Ryan et al, 2007;Song et al, 2010;Moreno-Pérez et al, 2011). Expression of these cDNA sequences in vivo using S. cerevisiae as a heterologous host resulted in the biosynthesis of both cis-and trans-isomers of plant characteristic 4-hydroxysphing-8-enines, which do not present in wild-type yeast cells (Sperling and Heinz, 2003).…”

A Higher Plant Δ8 Sphingolipid Desaturase with a Preference for (Z)-Isomer Formation Confers Aluminum Tolerance to Yeast and Plants

“…García-Maroto et al (2007) speculated that distinct D8 desaturases might have preferences for the cerebroside or glycosyl inositol phosphorylceramide groups of sphingolipids. The sphingobase phytosphinganine (t18:0) is a candidate substrate for S851 (Sperling et al, 2000).…”