Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue–specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Carotenoids are a large class of lipophilic molecules that give flowers, fruits, and vegetables bright red, orange, and yellow color (Yuan et al., 2015b). In plants, carotenoids and their derivatives are critically important for plant survival and development (Nisar et al., 2015; Rodriguez-Concepcion et al., 2018; Wurtzel, 2019). Carotenoids are vital for photoprotection and contribute to light harvesting for photosynthesis (Niyogi and Truong, 2013; Hashimoto et al., 2016). They serve as precursors for biosynthesis of phytohormones abscisic acid and strigolactones (Nambara and Marion-Poll, 2005; Al-Babili and Bouwmeester, 2015) and are attractants to pollinators and seed-dispensing animals for plant reproduction. Carotenoid derivatives also act as signals for plant development and stress responses (Havaux, 2014; Hou et al., 2016) and provide aroma and flavors for fruits and vegetables. In addition, carotenoids provide precursors for vitamin A synthesis and are dietary antioxidants to lower the risks of some chronic diseases in humans (Fraser and Bramley, 2004; Rodriguez-Concepcion et al., 2018). Their essential roles in plants and health-promoting properties in humans have led to intense efforts to understand and manipulate carotenoids in plants (Nisar et al., 2015; Yuan et al., 2015b; Giuliano, 2017; Rodriguez-Concepcion et al., 2018; Sun et al., 2018; Wurtzel, 2019).

Carotenoid biosynthesis occurs in plastids in plants (Sun et al., 2018). Phytoene synthase (PSY) catalyzes the “head-to-head” condensation of two molecules of geranylgeranyl diphosphate (GGPP) to form the first carotenoid phytoene, which represents the committed step in the carotenoid biosynthesis pathway. The subsequent phytoene desaturations and isomerizations produce red-colored lycopene. Lycopene is cyclized to form β,β- or β,ε-branch carotenes, which are further metabolized to xanthophylls (Moise et al., 2014).

As the first committed enzyme in carotenogenesis, PSY plays a key role in controlling metabolic flux into the pathway (Cazzonelli and Pogson, 2010). As such, PSY is used extensively for metabolic engineering of carotenoids in crops (Giuliano et al., 2008; Sun et al., 2018). For example, overexpression of PSY has been shown to achieve high levels of carotenoid production in tomato (Solanum lycopersicum) fruit (Fraser et al., 2007), canola (Brassica napus) seed (Shewmaker et al., 1999), potato (Solanum tuberosum) tuber (Ducreux et al., 2005), white carrot (Daucus carota ssp. sativus) root (Maass et al., 2009), and cassava (Manihot esculenta) root (Welsch et al., 2010). Overexpression of PSY also causes carotenoid overproduction in calli of many plant species (Paine et al., 2005; Maass et al., 2009; Cao et al., 2012; Mlalazi et al., 2012; Bai et al., 2014; Schaub et al., 2018). Moreover, PSY is used in combination with other carotenogenic genes for specific carotenoid and apocarotenoid enrichment in crops (Ye et al., 2000; Paine et al., 2005; Diretto et al., 2007; Zhu et al., 2008, 2018; Wang et al., 2014; Paul et al., 2017).

Phytoene synthase is normally found as a small family with up to three members in plants. Although Arabidopsis (Arabidopsis thaliana) contains only one PSY, there are two or three PSY isoforms in evolutionarily distant plants (Gallagher et al., 2004; Giorio et al., 2008; Li et al., 2008; Qin et al., 2011; Mlalazi et al., 2012; Fantini et al., 2013; Stauder et al., 2018; Ahrazem et al., 2019). PSY isozymes evolved following gene multiplication and exhibit functional divergence in response to developmental and physiological signals in plant tissues (Welsch et al., 2008; Ampomah-Dwamena et al., 2015; Stauder et al., 2018). However, it is not clear whether PSY isoforms have different enzyme activities and what amino acid residues in PSY sequences are critical for their activities.

The tomato genome contains three PSY genes (Sato et al., 2012). PSY1 is chromoplast specific and expresses in extremely high abundance in fruit at ripening stages (Giorio et al., 2008; Kachanovsky et al., 2012). PSY2 functions predominantly in chloroplast-containing tissues and does not contribute to carotenoid production in fruit (Fraser et al., 1999). PSY3 was recently found to express strongly during root interaction with symbiotic arbuscular mycorrhizal fungi for apocarotenoid/strigolactone formation (Stauder et al., 2018). PSY1 and PSY2 were generated by Solanum-specific whole-genome triplication (Sato et al., 2012). Their deduced protein sequences share high similarity beyond the putative transit peptide cleavage sites (Giorio et al., 2008). Although both PSY1 and PSY2 catalyze carotenogenesis, their biochemical properties are apparently different. Compared with PSY2, PSY1 was reported to be less dependent on Mn2+ as its divalent ion cofactor for activity, to exhibit pH optimum at more alkaline, and to have less affinity for GGPP (Fraser et al., 2000).

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