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.

Not only is oil palm by far the most productive oil crop, with annual yields often exceeding 4 tons/ha, but during fruit maturation, its mesocarp cells efficiently convert photosynthate to TAGs, which subsequently accumulate to levels as high as exceeding 90% of the tissue dry weight at maturity. In contrast, in the mesocarp of date palm, no TAGs are found. Instead, large amounts of sugars accumulate during maturation to levels as high as 50% of dry weight (9). For their study in PNAS, Bourgis et al. (9) obtained several million ESTs from developing fruit tissues of the two palm species. After gene annotation using Arabidopsis as a reference genome (3), expression levels of orthologous transcripts encoding lipid biosynthesis enzymes found in oil palm and date palm were compared. Sequence identities between the two palm species were generally higher than 90%, as expected for members of the same plant family. In a second approach, the authors monitored the levels of lipid

Bourgis et al. add a new chapter to our understanding of plant oil biosynthesis.

enzyme transcripts through oil palm mesocarp development including the transition to TAG production.

Plant lipid biosynthesis is compartmentalized differently from that of fungi and animals. De novo FAs biosynthesis occurs exclusively in the plastids, and TAG assembly is located in the endoplasmic reticulum (3). In oil palm fruit tissue, TAGs accumulate as oil droplets in the cytoplasm, as the electron micrographs of Bourgis et al. (9) show. This is in contrast to the membrane-bordered oil bodies of seeds (10). The compartmentation of the complete biosynthetic pathway necessitates that the photosynthate, supplied as sugar from the leaves, not only be imported into the mesocarp cells but also be transferred to the plastid lumen to become substrate for FA biosynthesis.

Most of the Action Is in the Plastid

All plant cells are autonomous in respect to acyl lipid biosynthesis, each supplying all their own membrane lipids. In principle, only one enzymatic step is needed to convert common membrane lipids like diacylglycerol or phosphatidyl choline to TAG. So, how then is the oil palm mesocarp cell programmed for the divergence of almost all photosynthate to lipid storage, when date palm cells store photosynthate simply as sugars? A priori one would predict that there must be an up-regulation of all of the pertinent components from FA biosynthesis, precursor supply, as well as lipid assembly. However, this is not what Bourgis et al. (9) find. Surprisingly, none of the ER enzymes of the so-called Kennedy pathway, including DGAT or PDAT, enzymes essential for TAG biosynthesis, were strongly up-regulated in oil palm. Most transcript levels were found to be the same; no or only very subtle increases were found during mesoderm development. Clearly, the enzymes known for this segment of the pathway appear to have an enormous capacity of adjusting to metabolic fluxes, possibly 100 times higher than in a general plant cell. In contrast, all transcripts encoding the 18 plastidial FA biosynthesis enzymes involved in the conversion of pyruvate to FAs were found significantly up-regulated in oil palm, 13 times on average, and the three members of the multisubunit FA synthase between 17 and 44 times. Somewhat more moderate but significant up-regulation of the same plastidial FA enzymes during oil palm mesoderm development were observed as well, corroborating the interspecies comparison. As the plastids of the oil palm mesoderm produce a large quantity of FA, are there any transcriptional signatures for photosynthate transport, and how is glycolysis affected? Again, most of the action appears to happen in the plastid; only marginal modulations were observed in the cytoplasm. Plastidial transporters for hexose, pentose, triose phosphate, and phosphoenolpyruvate were strongly up-regulated in oil palm, indicating increased capacity of carbon flow into the plastid, and several plastidial glycolytic enzymes were elevated as well. Photosynthate from the leaves arriving as sucrose can be cleaved by invertase or sucrose synthase, and in developing oilseeds, a switch from the former to the latter is observed during the transition to oil biosynthesis (11). It is interesting to note that, in oil palm mesoderm, 35 times more cell wall invertase than sucrose synthase transcripts were detected. Either the increase of sucrose synthase in seeds is correlated with oil biosynthesis but is not causal (12), or in mesoderm, alternative metabolic routes have evolved.

In summary, the differential analysis of the two palm mesocarp transcriptomes revealed the following picture: in oil palm, the arriving sucrose is cleaved to hexoses, and intermediates are transported to the plastid, where glycolysis and FA synthesis are massively up-regulated. The large flux of de novo FAs is then channeled to the endoplasmic reticulum, where “housekeeping” levels of the Kennedy pathway and associated enzymes are sufficient to assemble the TAG, which simply accumulates as oil droplets in the cytoplasm.

A WRINKLED1 for More Oil?

The EST data not only revealed a very clear picture of transcriptional modulation of enzymes, enabling the upscaling of acyl lipid biosynthesis, but shed some new light into the regulation of lipid gene expression as well. Of nearly 1,000 putative transcription factors detected, only a handful were found up-regulated during oil palm maturation and at higher levels than found in date palm. One of these candidates encodes a polypeptide with high similarity to the Arabidopsis WRINKLED1 transcription factor (WRI1). A mutant of WRI1 had been discovered through an Arabidopsis low-oil seed selection, which accumulated only 20% of normal TAG levels in its seeds (13). The respective gene was identified as encoding an APETALA2-type transcription factor, and the mutant rendered developing embryos unable to efficiently produce FAs (14). WRI1 appears to be regulated by LEC1 and LEC2, key transcriptional regulators of seed maturation, and it effects the signal from LEC1, LEC2, and other factors (15) to the transcriptional up-regulation of at least 10 FA biosynthesis genes, hence it is a metabolic pathway “master switch” (16). Indeed, ectopic expression of Arabidopsis WRI1 has been shown to induce FA biosynthesis and TAG accumulation in seedlings of Arabidopsis (14). As it is up-regulated in maturing oil palm mesoderm, it appears that a WRI1 isoform was recruited during oil palm evolution to act in the maturing fruit tissue. Interestingly, no obvious orthologues to Arabidopsis LEC1, LEC2, and other transcription factors known to act on WRI1 in the seed were found in oil palm mesoderm. Likely, WRI1 in palm mesoderm responds to a very different network of transcription factors.

By delivering a comprehensive framework of carbon partitioning in an oil-producing, nonseed tissue, Bourgis et al. (9), add a new chapter to our understanding of plant oil biosynthesis. The results demonstrate some of the principal mechanisms so often observed in evolution, in which, out of established networks of enzymes and transcription factors, through some reshuffling of modules, a new system is built. There are other species with oil-bearing mesocarp, like olive and avocado (17). Does carbon partitioning to TAGs in these likely independently evolved high-oil tissues follow the same principles as found in oil palm? Hopefully, such questions will be answered soon. Bourgis et al. (9) significantly add to our knowledge about carbon partitioning to oil, which might further the advance to the holy grail of engineering increased oil content in plants for food, feed, and energy.