Keeping an Eye on Lutein Stability

Carotenoid pigments not only produce the vibrant yellows and oranges of flowers, fruits, and autumn leaves but also are important in both plant and human health. They act both as accessory pigments in photosynthetic light harvesting and as photoprotectants that absorb excess energy during photosynthesis. β-Carotene is a well-known dietary carotenoid that, when converted into retinal, is important for human vision (Rodriguez-Concepcion et al., 2018).

Lutein (from the Latin luteus for yellow) is a carotenoid found in the central area of the retina known as the macula, an area important for color vision (Johnson, 2014). Lutein alleviates macular degeneration and improves vision, perhaps by absorbing excess blue and UV light that enters the eye. It is the primary carotenoid in mature wheat grains. In diets poor in fresh fruits and vegetables, cereal grains can be the primary source of lutein. Esterification of lutein with fatty acids, such as linoleic or palmitic acid, stabilizes lutein in wheat grains. Despite much recent progress in carotenoid biochemistry and metabolism (Nisar et al., 2015; Zhai et al., 2016), the genetic and enzymatic mechanisms responsible for lutein esterification have remained elusive.

Using both a wheat cultivar that does not produce lutein esters and a substitution line in which its chromosome 7A was replaced by chromosome 7D from a line that accumulates lutein esters, Watkins et al. (2019) showed that this chromosome confers the ability to esterify lutein in wheat grains. On this chromosome, they then identified and isolated xanthophyll acyltransferase (Xat) and showed that XAT was necessary and sufficient for lutein esterification in wheat grains. Six independent TILLING (targeted induced local lesions in genomes) lines mutated in Xat did not make lutein esters.

To verify its lutein esterification activity in vitro, recombinant XAT was produced in Escherichia coli and used in assays with lipids from a high-lutein line lacking both XAT and lutein esters. Addition of recombinant XAT resulted in production of several types of lutein esters. Because wheat XAT contains the conserved Gly-Asp-Ser-Leu (GDSL) motif found in a number of esterases/lipases, and that Ser-37 in this motif is important in catalysis, the authors mutated the Ser to Ala. Lutein esterification was abolished, confirming a vital catalytic role for Ser-37.

Analysis of the XAT esterification reaction in vitro with a variety of acyl donors and lutein as acceptor showed triacylglycerides to be the preferred donor. More detailed analysis allowed the authors to determine the K_m for lutein to be ∼60 μM and revealed that XAT has both esterase and lipase activity.

In situ hybridization with labeled riboprobes showed that Xat transcripts were abundant in endosperm and aleurone. To determine the subcellular localization of XAT, the authors produced fusions of the predicted signal peptide (SP) of XAT with mCherry with or without the XAT coding sequence and expressed the constructs in Nicotiana benthamiana leaves. Surprisingly, because carotenoids are found primarily in plastids, XAT localized to the apoplastic region just outside the plasma membranes of adjacent cells (see figure).

XAT Is Found in the Apoplast.

This fluorescence image of mCherry (magenta) and a plasma membrane marker (green) shows that SP-mCherry-XAT localizes to the apoplast when expressed in N. benthamianaleaves. Bar = 20 μm.  (Reprinted from Watkins et al. [2019], Figure 6.)

The authors propose that XAT and its carotenoid substrates (such as lutein) are separated spatially during grain maturation. During grain maturation, programmed cell death allows enzyme and substrate to react, stabilizing lutein in the mature grain. To further investigate lutein esterification in vivo, they used rice embryonic callus as a model system, with an eye toward improving the nutritional value of cereals. For example, introgression of Xat from bread wheat (Triticum aestivum) into durum wheat (Triticum turgidum ssp durum) to stabilize its lutein could provide us with healthier, more attractive, and higher quality pasta, simultaneously satisfying nutritional, aesthetic, and industrial goals.