Toni Voelker

DGAT2 Is a New Diacylglycerol Acyltransferase Gene Family PURIFICATION, CLONING, AND EXPRESSION IN INSECT CELLS OF TWO POLYPEPTIDES FROM MORTIERELLA RAMANNIANA WITH DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY

Acyl CoA:diacylgycerol acyltransferase (EC2.3.1.20; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity, were isolated from the lipid bodies of the oleaginous fungusMortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals, which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer to the M. ramanniana genes asMrDGAT2A and MrDGAT2B.

Posttranscriptional gene silencing in nuclei

In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.

Secrets of palm oil biosynthesis revealed

Globally, vegetable oils are harvested from a handful of oil crops at an annual rate exceeding 100 million tons (1). Most oils are used for human or animal consumption, although a minor fraction is derivatized to oleochemicals. More recently, an increasing amount of vegetable oil is being diverted to the production of biodiesel [i.e., fatty acid (FA) methylesters], and is an attractive feedstock for the so-called “drop-in” biofuels of the future, further increasing the demand on this commodity (2). Because of the commercial importance of vegetable oils, during the past 30 y, the biosynthesis of FAs and their derived acyl lipids, especially plant triacylglycerols (TAGs), has attracted a tremendous amount of scientific interest, resulting in the identification and characterization of the functions of most of the enzymes involved in their production and sequestration (3). In addition, researchers have attempted to understand the quantitative regulation of biosynthesis with the ultimate goal of increasing oil production in crops. Practically all investigations in this field have been conducted in developing embryos of oilseeds, most notably in Arabidopsis. As cDNA sequences of enzymes considered “rate-limiting” became available in the mid-1990s, increasing oil production in plants has become the “holy grail” of plant FA biochemistry (4, 5). However, even though there are more than a dozen reports of marginal increases of seed oil through genetic manipulation, channeling carbon intermediates to TAGs has had very limited success, and no high-oil engineered crop is on the market (6, 7). Only very recently a comprehensive analysis of oil palm mesoderm during fruit ripening was published by Tranbarger et al. (8). With this investigation, Tranberger et al. stepped “outside the box” (i.e., the seed) and delivered a first detailed insight into the compositional, hormonal, and transcriptional changes during the different phases of oil palm fruit development and compared their finding to the established knowledge of oilseed development. While comparing the transcriptomes and metabolomes of developing oil accumulating mesoderm of oil palm (Elaeis guineensis) and sugar accumulating mesoderm of date palm (Phoenix dactylifera), Bourgis et al. (9) follow a strategy that enables them to differentiate metabolic determinants of the respective tissues from other developmental phenomena.