Uncovering the secrets to vibrant flowers: the role of carotenoid esters and their interaction with plastoglobules in plant pigmentation

Abstract

Carotenoids, once biosynthesised, participate in a range of processes within plants including serving as precursors for the biosynthesis of multiple hormones, acting as signalling molecules and apocarotenoid aroma compounds, as well as playing roles in the stabilisation of photosystems and acting as antioxidants. These functions contribute to a high turnover rate of carotenoids within leaves (Beisel et al., 2010). Another facet of carotenoids is in attracting insects and animals to facilitate successful reproduction and seed dispersal due to their vibrant colours and presence in fruits and flowers. However, this process relies upon their stable storage in the plastids of non-photosynthetic tissue. The quantity and composition of carotenoids is highly species and variety specific, but generally, the total carotenoid content correlates to the colour intensity in these organs. The esterification of xanthophylls (oxygenated carotenoids) to fatty acids positively influences total carotenoid accumulation by enhancing their packaging into specialised structures within plastids, called plastoglobules. Esterification also likely protects xanthophylls from catabolism through steric hindrance of enzymes that catalyse carotenoid cleavage, such as the carotenoid cleavage dioxygenases and the 9-cis-epoxycarotenoid dioxygenases, although this remains to be demonstrated. Despite the positive influence on total carotenoid accumulation, we are only beginning to understand the molecular mechanisms involved in xanthophyll ester production.

Using a comprehensive set of experiments, Li et al. unravel the genetic basis of esterification in rapeseed flowers. Through a combination of map-based cloning, loss-of-function studies using CRISPR/Cas9 technology and genetic complementation, two homologous genes from the esterase/lipase/thioesterase (ELT) family of acyltransferases were identified that function redundantly to direct petal colour formation and are annotated as xanthophyll esterases. The authors used liquid chromatography coupled with UV/vis spectroscopy and high-resolution mass spectroscopy to identify individual xanthophyll ester species, an undertaking which is notoriously difficult (Mercadante et al., 2017). Interestingly, in white petals of both a naturally occurring cultivar and the xanthophyll esterase double knockout line, pcs, not only are esterified xanthophylls absent, but total carotenoid content is greatly reduced, signifying that in addition to biosynthesis, the stable storage and protection of carotenoids from turnover is required to produce the vivid yellow petal phenotype. The authors further probed the metabolome and transcriptome of the xanthophyll esterase double mutant line pcs and discovered that in the white petals of the mutant, metabolic flux is redirected to lipid metabolism and storage. They also observed no change in the expression of carotenoid biosynthesis genes but saw an upregulation of several lipoxygenase (LOX) genes as well as carotenoid cleavage dioxygenase 1 (CCD1) enzymes, which may participate in the decay of carotenoids and further exacerbate the white petal phenotype.

During the transition from chloroplasts to chromoplasts, plastids undergo extensive remodelling, which includes disassembly of thylakoid membranes alongside the proliferation of specialised carotenoid-storage structures called plastoglobules. Plastoglobules consist of a core of neutral lipids surrounded by a protein-studded phospholipid monolayer with fibrillin proteins playing a crucial structural role in the outer shell (Rottet et al., 2015; Fig. 1). Plastoglobules are present in multiple types of plastids (chromoplasts, chloroplasts, etioplasts) where they host specific metabolic functions in response to developmental cues, such as flowering and senescence, as well as various stresses such as high light or nitrogen starvation (van Wijk & Kessler, 2017). In chromoplasts, the core of the plastoglobules accumulate carotenoids both via de novo biosynthesis and remobilisation through the thylakoid and plastid envelop membranes. Li et al., therefore, used a reverse genetic approach employing CRISPR/Cas9 technology to knockout individual fibrillin candidates, which led to the identification of FBN1b as being crucial for plastoglobule formation. Similar to the xanthophyll esterase double mutant line pcs, the fbn1b edited knockout line also displays white flower colouration, and whilst it does produce a small amount of xanthophyll esters, the total xanthophyll content is greatly reduced.

Given that abolishing xanthophyll esterification and plastoglobule formation both lead to white petal phenotypes, questions arise as to how these processes influence ultrastructural reorganisation that occurs both in the chloroplast to chromoplast transition during petal development and during senescence. Li et al. addressed these questions by performing transmission electron microscopy on the yellow petal (wild type) and the white petal xanthophyll esterase and fbn1b knockout lines over a developmental time course. Li et al. observed that the size and number of plastoglobules were significantly reduced in the xanthophyll esterase double mutant line pcs at all-time points, whereas the fbn1b mutants were unable to produce plastoglobules and instead produced large oil body-like structures. In late petal development, when petals are beginning to dehydrate and wilt, chromoplasts in yellow petals maintained their integrity, whereas in both the xanthophyll esterase and fbn1b, white petal lines plastid membranes displayed visible damage as well as a significant reduction in the number of plastids per cell. The authors speculated if this was due to cellular senescence triggered by reactive oxygen species (ROS) accumulation, which is supported by higher levels of singlet oxygen in white petals.

Taken together, experiments conducted by Li et al. point towards an integrated model of yellow petal colouration, which is regulated by both xanthophyll esterification and plastoglobule formation. Esterification of xanthophylls allows for enhanced accumulation and packaging of pigments into plastoglobules, which in turn can only be formed with a functional copy of the FBN1b protein to support the outer shell (Fig. 1). Without the FBN1b protein and thereby plastoglobule formation, the xanthophylls, both free- and esterified, accumulate to a greatly reduced extent and are also more susceptible to degradative enzymes including CCD1 and LOX, both of which are upregulated in white flowers. With functional copies of FBN1b and proper plastoglobule formation, the enhanced accumulation of total xanthophylls also provides greater antioxidant capacity that attenuates the cellular accumulation of damaging ROS. This, in turn, protects the plastoglobule membrane from damage and limits leakage of xanthophylls into the cellular environment, where they would be exposed to enzymes involved in their breakdown.

Results from Li et al. determine that the xanthophyll esterases in rapeseed petals are part of the ELT acyltransferase family. This contrasts with the previously identified xanthophyll acyltransferase characterised from wheat grain (Watkins et al., 2019) that was recruited from the Gly-Asp-Ser-Leu esterase/lipase family and showed different preferences for both xanthophyll and fatty acid substrates. This suggests that xanthophyll esterification has evolved independently at least twice in plants. Esterification also plays an important role in the accumulation of the commercially important ketocarotenoid, astaxanthin, in microalgae (Chen et al., 2015). Astaxanthin is used in dietary supplements, cosmetics, and food products as a natural colorant and functional ingredient. Whilst the fibrillin protein family is known to be conserved amongst algae and cyanobacteria (van Wijk & Kessler, 2017), the genetic mechanisms of xanthophyll esterification remain unknown, yet may provide tools to ask evolutionary questions regarding carotenoid accumulation and storage.

Despite significant progress in understanding xanthophyll esterification in floral tissue and cereal grains, esterification mechanisms in other important plant tissues such as fruits (e.g. bananas, oranges, papaya and peppers) and tubers (e.g. potatoes) are still unknown. The colour of the edible flesh of these crops varies widely amongst cultivars and is an important quality trait influencing consumer preference. Potatoes and bananas are amongst some of the most important staple crops in the world, providing significant caloric intake for millions of people around the world. In potatoes, the yellow colour is given by xanthophylls, with esterification directly correlating to their accumulation (Fernandez-Orozco et al., 2013). Investigation of esterification in these plants may lead to more targets for breeding or gene editing programmes. Additionally, oranges, papayas and peppers contain chromoplasts, similar to rapeseed flowers, whereas potato tubers and banana flesh possess starch-containing amyloplasts, similar to wheat grain. Further research could also explore whether xanthophyll esterification and storage mechanisms differ across plastid types.