This has been verified for A. thaliana DGAT1 and DGAT2. Here we report the functional characterization of A. thaliana DGAT2 in the yeast mutant strain H1246 which is unable to accumulate neutral lipids. We demonstrate that yeast codon usage limits protein expression in this system and that expression of codon-optimized DGAT2 can restore TAG synthesis by functional complementation. A. thaliana DGAT1 was used as a positive control. We then show that the two DGAT proteins are associated with LDs. Both enzymes exhibit contrasted substrate specificity and induce squalene accumulation in LDs. The specialization of DGAT2 observed in the present study is consistent with these last findings. Similarly, transient heterologous expression of DGAT2 in tobacco leaves leads to the accumulation of TAG containing unsaturated FA revealing possible expression under the subtle control of tissue specific factors. Thus, very long chain FA or polyunsaturated FA are potential acyl donors found in A. thaliana seeds and N. benthamiana leaves, but absent in S. cerevisiae. It is therefore difficult to compare enzyme specificity and velocity as reflected by lipid accumulation for DGATs expressed in heterologous systems. Overall, the results strongly suggest that these enzymes show specificity toward different acyl donors. Our confocal microscopy investigations confirmed the colocalization of DGAT-GFP and neutral lipids. The punctuate structures observed after a short induction were typical of LDs, Gefitinib however Nile Red staining was more diffuse after longer induction times. The lipids which accumulated in DGAT expressing yeast were different from the WT strain and lacked sterol esters. As previously suggested by Czabany, lipid composition affects LD protein content and as a consequence, the interactions between LDs and their environment could be modified. Nevertheless the objects purified from the yeast which had been induced for 18 h still had a typical LD hydrodynamic diameter. Thus it could be that close association of numerous LDs rendered individual droplets undistinguishable in vivo using light microscopy, explaining the shape of structures observed after 18 h induction The association of DGAT1 with LDs and the ER was previously demonstrated by Bouvier-Nave´ et al. DGAT2 localization to microsomes was deduced from in vitro assays by Zhou et al., however its presence in other compartments cannot be excluded. Recent results by Kwiatkowska et al. tend to suggest multiple localizations of DGAT2 in germinated A. thaliana seeds. These last experiments were based on the use of antibodies directed against human DGAT2, and the crossreactivity between plant proteins and human antibodies needs to be confirmed. Taken together, these reports and our present findings appear to support the general model proposing that TAG synthesis occurs at the ER with LDs budding from the ER. Research using transgenic zebrafish lines has greatly contributed to our understanding of vertebrate biology.