A Neighboring Aromatic-Aromatic Amino Acid Combination Governs Activity Divergence between Tomato Phytoene Synthases

Abstract

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major ratelimiting 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 (

Characterization of Cauliflower OR Mutant Variants

“…The lycopene produced in E. coli can be quantified photometrically, which provides a convenient read-out corresponding to PSY enzymatic activity as phytoene is readily and fully converted by CrtI (Camagna et al, 2019). This system was successfully used to determine PSY activity differences caused by single nucleotide or amino acid polymorphisms (Welsch et al, 2010;Mlalazi et al, 2012;Cao et al, 2019).…”

“…PSY was also shown to interact with the upstream substrate-delivering enzyme GGPP synthase (GGPS) and acts as a constituent of metabolons containing up-and downstream enzymes to provide an additional possibility for regulation at the level of protein-protein interaction (Maudinas et al, 1977;Fraser et al, 2000;Ruiz-Sola et al, 2016;Camagna et al, 2019). Its evolutionary conserved features for activity across various organisms were recently analyzed (Cao et al, 2019). As PSY protein level is crucial for overall pathway activity and carotenoid amounts, a tight control of PSY proteostasis is expected.…”

Carotenoid biofortification in crop plants: citius, altius, fortius

“…Activity studies and site directed mutagenesis confirmed the role of this structural element [187]. Additional amino acid differences were found to be responsible for large differences in the activities of the daffodil and maize PSY enzymes, used to engineer "Golden rice" plants with different provitamin A contents [187,188]. The identification of highly-efficient PSY alleles paves the way for generating them in other tissues or plant species by employing modern gene editing tools, such as CRISPR mediated targeted base editing [189].…”

“…In cassava, the allelic variation (Ala > Asp mutation) in PSY leads to enhanced enzymatic activity and, hence, to increased carotenoid accumulation in yellow-rooted cassava varieties with increased provitamin A content [186]. Recently, examination of two tomato PSY isoforms (PSY1 and PSY2) identified a neighboring aromatic-aromatic combination in their core structure, which is conserved in PSY enzymes from different species but absent in the fruit specific and enzymatically less efficient PSY1 [187]. Activity studies and site directed mutagenesis confirmed the role of this structural element [187].…”

“…Recently, examination of two tomato PSY isoforms (PSY1 and PSY2) identified a neighboring aromatic-aromatic combination in their core structure, which is conserved in PSY enzymes from different species but absent in the fruit specific and enzymatically less efficient PSY1 [187]. Activity studies and site directed mutagenesis confirmed the role of this structural element [187]. Additional amino acid differences were found to be responsible for large differences in the activities of the daffodil and maize PSY enzymes, used to engineer "Golden rice" plants with different provitamin A contents [187,188].…”