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. 2019 Jun 20;180(4):1988–2003. doi: 10.1104/pp.19.00384

A Neighboring Aromatic-Aromatic Amino Acid Combination Governs Activity Divergence between Tomato Phytoene Synthases1,[OPEN]

Hongbo Cao a,b,c, Hongmei Luo d,e, Hui Yuan a,d, Mohamed A Eissa d,f, Theodore W Thannhauser a, Ralf Welsch g, Yu-Jin Hao c, Lailiang Cheng h, Li Li a,d,2,3
PMCID: PMC6670109  PMID: 31221734

A lack of neighboring aromatic-aromatic amino acid combination in one of the PSY core structures is responsible for the weak carotenogenic activity of tomato fruit-specific PSY1.

Abstract

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).

In this study, we took advantage of the model plant and bacterial systems to investigate two tomato PSY isozymes for their respective activities. We demonstrate that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue–specific PSY2. We also uncovered the critical amino acid residues important for PSY activity and enzyme evolution.

RESULTS

Tomato Fruit-Specific PSY1 Exhibits Significantly Weaker Carotenogenic Activity Than Green Tissue–Specific PSY2

The tomato PSY family evolved in such a way that PSY1 functions predominantly in fruit, whereas PSY2 functions mainly in green tissues (Fraser et al., 1994; Giorio et al., 2008). Tomato PSY1 and PSY2 share about 78% protein sequence similarity, with the main sequence differences in the transit peptide regions (Fig. 1A), as reported previously by Giorio et al. (2008). Subcellular localization analysis of the PSY–GFP fusion proteins revealed that both PSY1 and PSY2 were targeted to plastids (Fig. 1B). Using SWISS-MODEL (Waterhouse et al., 2018), the predicted three-dimensional (3D) models of the mature PSY1 and PSY2 proteins also showed high similarity to each other (Fig. 1C).

Figure 1.

Figure 1.

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Tomato PSY1 and PSY2 protein sequence comparison, subcellular localization, and predicted 3D protein structures. A, Alignment of protein sequences of tomato PSY1 (GenBank ID: P08196) and PSY2 (GenBank ID: A9Q2P8). White inverted triangle indicates the predicted transit peptide cleavage site at residue 62 of PSY1 and 86 of PSY2. Black inverted triangle points to residue 138 of PSY2. Red inverted triangles indicate the sites and the residue numbers of tomato PSY1 for site-directed mutagenesis (SDM) analysis. Blue shading indicates the homology levels at 100%. B, Subcellular localization of tomato PSY1 and PSY2 in Nicotiana benthamiana leaves. The images show plastid localizations. Left, GFP green fluorescence. Middle, Chlorophyll red fluorescence. Right, Merge of GFP and chlorophyll signals in bright field background. Scale bars = 10 μm. C, Overview of the predicted 3D protein structures of mature tomato PSY1 and PSY2 based on SWISS-MODEL (Waterhouse et al., 2018).

To compare the carotenogenic activities of PSY isoforms, we overexpressed tomato PSY1 and PSY2, respectively, in Arabidopsis. An effective callus system routinely used to monitor carotenogenesis (Cao et al., 2012; Bai et al., 2014; Yuan et al., 2015a; Schaub et al., 2018) was utilized to assess their activities. A number of homozygous PSY1 and PSY2 overexpression lines were generated. Two independent homozygous PSY1 lines (nos. 17 and 25) and two PSY2 lines (nos. 16 and 23) were selected and used for callus induction. As shown in Figure 2A, the PSY gene expression in the callus tissue was comparable among these PSY1 and PSY2 overexpression lines. Similar PSY protein levels were also observed in the calli of these two PSY1 lines in comparison with the two PSY2 lines (Fig. 2B). Overexpression of both PSY1 and PSY2 caused the formation of orange calli (Fig. 2C). Noticeably, the color of PSY1 transgenic calli was less intense or less dark orange than that of PSY2 transgenic calli (Fig. 2C).

Figure 2.

Figure 2.

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Tomato PSY1 and PSY2 show different capacities in promoting carotenoid accumulation in transgenic Arabidopsis. A, Reverse transcription quantitative PCR (RT-qPCR) analysis of the relative expression of PSY1 and PSY2 transgenes in calli of transgenic Arabidopsis (nos. 17 and 25, PSY1 overexpression lines; nos. 16 and 23, PSY2 overexpression lines). Because no tomato PSY transcript was present in nontransgenic plants, the expression of PSY1 overexpression line no. 17 was set to 1. Values are mean ± sd of three biological replicates. B, Immunoblot analysis of tomato PSY1 and PSY2 protein levels in the calli of Arabidopsis overexpression lines using anti-AtPSY antibody. Ponceau S staining shows protein loading. C, Representative callus phenotype of the PSY1 and PSY2 overexpression lines. Scale bars = 0.5 cm. D, Callus carotenoid levels and profiles of the PSY1 and PSY2 overexpression lines. The individual carotenoids are as indicated. Values are mean ± sd of three biological replicates. E. Phytoene levels in norflurazon-treated leaves of the PSY1 and PSY2 overexpression lines. Values are mean ± sd of three biological repeats. *P < 0.05 and **P < 0.01 indicate significant difference based on the Student–Newman–Keuls multiple range test. FW, fresh weight.

To confirm that the observed color difference was due to different levels of carotenoid accumulation, carotenoids from calli of these lines were extracted and analyzed by ultraPerformance convergence chromatography (UPC2). The calli from these transgenic lines contained primarily β-carotene with some levels of phytoene, phytofluence, α-carotene, and lutein (Fig. 2D). Whereas α-carotene and lutein accumulated similarly in the calli of both PSY1 and PSY2 lines, the levels of β-carotene, phytoene, and phytofluene were lower in PSY1 than PSY2. The total carotenoid levels were significantly lower in calli of PSY1 than PSY2 lines, showing a decrease up to 2-fold (Fig. 2D). The transcript levels of 19 endogenous carotenoid metabolic or related genes in calli of PSY1 and PSY2 overexpression lines were examined by RT-qPCR. Similar expression of all those endogenous genes was observed between the PSY1 and PSY2 overexpression lines (Supplemental Fig. S1). These results indicate that the lower carotenoid levels in the PSY1 lines were not due to decreased carotenogenic gene expression.

Norflurazon (NFZ) is a phytoene desaturase inhibitor that is commonly used to determine in vivo PSY activity by measuring phytoene accumulation in plant tissues (Rodríguez-Villalón et al., 2009; Zhou et al., 2015). To further investigate the respective activities of tomato PSY1 and PSY2 in carotenogenesis, NFZ treatment was also carried out in leaf tissues of these four transgenic lines. Consistent with the data from callus samples, significantly lower levels of phytoene were detected in the PSY1 lines compared with the PSY2 lines (Fig. 2E). These findings demonstrate that the fruit-specific PSY1 had weaker carotenogenic activity than the green tissue–specific PSY2.

Engineered Bacterial Cells with PSY1 Produce Less Lycopene Than PSY2

In plants, the subcellular location, membrane topology, and association with other proteins all influence PSY enzymatic properties and activities (Shumskaya et al., 2012; Zhou et al., 2015; Welsch et al., 2018). An engineered Escherichia coli system is a very useful tool to study carotenogenesis, which has provided abundant insights into the action of carotenoid enzymes (Misawa et al., 1990; Cunningham and Gantt, 1998; Welsch et al., 2010; Mlalazi et al., 2012; Camagna et al., 2019; Nogueira et al., 2019). Tomato PSY1 showed weaker carotenogenic activity than PSY2 in those transgenic Arabidopsis lines (Fig. 2). To see if this was the case in bacteria, a BL21 E. coli–based engineered system that expressed Arabidopsis GGPS11 and bacterial carotene desaturase (CrtI) was used to express PSY1 and PSY2 without transit-peptide coding sequences.

Coexpression of GGPS11, PSY, and CrtI in the E. coli system leads to the production of lycopene (Camagna et al., 2019). As shown in Figure 3A, introducing tomato PSY1 or PSY2 into the engineered E. coli containing GGPS11 and CrtI resulted in the formation of lycopene with red color cells. Consistent with the results obtained in plant tissues (Fig. 2), the E. coli cells expressing PSY2 exhibited darker red color than those harboring PSY1 (Fig. 3A). Quantification of lycopene levels by spectrophotometry revealed an over 3-fold increase in the E. coli cells expressing PSY2 in comparison to expressing PSY1 (Fig. 3B). Immunoblot analysis revealed that those cells contained similar levels of PSY proteins (Fig. 3C). Because the expression levels of GGPS and CrtI in E. coli could also affect lycopene production, we checked GGPS protein levels and CrtI transcript abundance. GGPS and CrtI expression levels were found to be comparable in the E. coli cells expressing PSY1 and PSY2 (Fig. 3, C and D). Taken together, these data show that tomato PSY2 had stronger ability than PSY1 to promote lycopene production, which was not associated with increased expression of either PSY or GGPS and CrtI. This result was consistent with that obtained in transgenic Arabidopsis showing that the fruit-specific PSY1 exhibited weaker carotenogenic activity.

Figure 3.

Figure 3.

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Different capacity of carotenogenesis in the engineered E. coli cells expressing PSY1 or PSY2. A, Typical pellet color of the engineered E. coli cells harboring PSY1 or PSY2. Each tube contains cells from an individual colony. B, Relative lycopene level in the cells expressing PSY1 or PSY2. Values are mean ± sd of three biological repeats. ** Significant difference (P < 0.01) based on the Student–Newman–Keuls multiple range test. C, Immunoblot analysis of PSY and GGPS protein levels in the E. coli cells inoculated from two individual colonies per construct. Anti-AtPSY and anti-GGPPS11 antibodies were used. Ponceau S staining shows protein loading. D, Relative expression levels of the CrtI gene in the E. coli cells harboring PSY1 or PSY2. Two individual colonies were inoculated from each construct. Values are the average ± sd of three repeats.

Two Amino Acid Residues Are Associated with the Functional Variance between PSY1 and PSY2

Analysis of tomato PSY protein sequences revealed that the amino acid residues 75-405 in PSY1 and 92-430 in PSY2 belong to the conserved domain of isoprenoid biosynthesis C1 superfamily (Fig. 4A). The conserved domain contains some predicted key sites involved in substrate and substrate-Mg2+ binding, the active site lid residues, catalytic residues, and Asp-rich regions (Fig. 4A). There are only 45 amino acid residue differences between PSY1 and PSY2 in the conserved domain, and most are close to the N terminus (Fig. 1A). The residue variations were not located in those key sites, although several were close, such as residues Asn-136, Phe-354, and Val-361 in PSY1 (Figs. 1A and 4A). To see which residue variations were likely responsible for the high PSY2 activity, we used the engineered bacterial system to express a truncated PSY2 containing amino acids 138 to 438 (PSY2△138), which excluded most of the different residues close to the N terminus in the conserved domain (22/45; Fig. 1A). Interestingly, PSY2△138 remained similarly high activity in promoting same amount of lycopene production as the full-length PSY2 (PSY2△0) and the transit peptide-removed PSY2 (PSY2△86) in E. coli (Fig. 4B). These results indicated that the residues close to the N terminus were not important for the activity difference between PSY1 and PSY2.

Figure 4.

Figure 4.

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Two amino acids are important for activity difference between PSY1 and PSY2. A, Prediction of the conserved domain and key sites of PSY1 and PSY2 proteins via Conserved Domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The red inverted triangles indicate the sites and the residue numbers of tomato PSY1 for SDM analysis. The numbers above lines for PSY1 and PSY2 indicate amino acid sequencing positions. The black arrows above thin lines in the conserved domain indicate some predicted key sites. B, Functional analysis of truncated PSY2 in the engineered E. coli system. Representative cell pellet color with each tube inoculated from a single colony (top) and relative lycopene level (bottom) in the cells expressing various PSY2 constructs. △86 was set at 1. △0, full-length PSY2 with transit peptide; △86, mature PSY2 without transit peptide; △138, truncated PSY2 that excluded most of the different residues close to the N terminus in the conserved domain. C, SDM analysis of PSY1 in the engineered E. coli system. Representative cell pellet color from three individual colonies per construct (top) and PSY immunoblot analysis with anti-AtPSY antibody (bottom). △ctp, without transit peptide. N136Y and A176D, mutated PSY1. D. Relative lycopene levels in E. coli cells containing various SDM forms of PSY1. PSY1△ctp was set to 1. Values are mean ± sd of three biological repeats. *P < 0.05 and **P < 0.01 indicate significant difference based on the Student–Newman–Keuls multiple range test.

Five residue sites between 138 to 438 of PSY2 (Fig. 1A) were selected to be potentially associated with the functional variance between PSY1 and PSY2. The selection was partially based on the PolyPhen-2 analysis (http://genetics.bwh.harvard.edu/pph2/), which is an online software tool for predicting substitution tolerance in proteins (Adzhubei et al., 2010). In addition, we considered proximity to the conserved key domains. SDM was carried out to specifically change one of the selected amino acids of PSY1 to the corresponding amino acid of PSY2 in each construct. The selected amino acid residues in PSY1 included Asn-136, Gly-198, Phe-354, Val-361, and Ala-392 (Figs. 1A and 4A). Moreover, the residue Ala-176 in PSY1 was selected as a positive control because the corresponding Ala to Asp substitution in cassava has been shown to improve PSY activity in the bacterial system and in cassava root (Welsch et al., 2010). While the mutant forms of F354W, V361L, and A392T of PSY1 showed minimal effects on bacterial cell color and lycopene accumulation, N136Y and G198A greatly enhanced cell color and total lycopene levels (Fig. 4, C and D). PSY1(N136Y) exhibited the largest effect on promoting lycopene production, with an approximately 3-fold increase, and PSY1(G198A) caused a 1.5-fold increase (Fig. 4D). The combination of N136Y and G198A showed similar improvement of PSY1 activity compared with the single substitution of N136Y, and the mutant PSY1(N136Y) was as effective as PSY2 in promoting lycopene formation (Supplemental Fig. S2). As expected, the positive control PSY1(A176D) also promoted a 1.8-fold increase of lycopene production in comparison with PSY1 (Fig. 4D).

To see whether the increased lycopene production was caused by enhanced PSY expression, we extracted proteins from the bacterial cells expressing PSY1, PSY1(N136Y), and PSY1(A176D) and performed immunoblot analysis using anti-PSY antibody (Zhou et al., 2015). The analysis revealed that the PSY protein levels were similar (Fig. 4C) and that the increased lycopene production was not due to more PSY proteins. In addition, examination of the expression of GGPS and CrtI also revealed that the increased lycopene production in PSY1(N136Y) and PSY1(A176D) was not due to more GGPS and CrtI expression either (Supplemental Fig. S3A). These results indicated that the two amino acid residue variations of Asn-136 and Gly-198 in PSY1, particularly Asn-136, were crucial for the activity difference between PSY1 and PSY2.

Neighboring Aromatic–Aromatic Combination Significantly Improves PSY Activity

The experiments described identified amino acid variation at the site of Asn-136PSY1/Tyr-160PSY2 being important for the activity difference between tomato PSY1 and PSY2. To understand how such amino acid variation affected PSY activity, we explored the protein 3D structures by homology modeling (Waterhouse et al., 2018). Although PSY crystal structures are not available, the PSY proteins contain conserved core structures such as the hydrophobic flap and Asp-rich motifs of human squalene synthases (SQS) and bacterial dehydrosqualene synthetases (CrtM; Fig. 5A; Supplemental Fig. S4A; Shumskaya et al., 2012). Both SQS and CrtM have known crystal structures (Pandit et al., 2000; Lin et al., 2010). Similar to PSY that catalyzes head-to-head condensation of GGPP to form phytoene, SQS and CrtM catalyze head-to-head condensation of two molecules of farnesyl pyrophosphate to form squalene and dehydrosqualene, respectively. Based on close sequence identities and similarities (Supplemental Fig. S4B), PSY1 and PSY2 3D structures were modeled according to CrtM (Protein Data Bank ID:5IYS) through SWISS-MODEL (Waterhouse et al., 2018).

Figure 5.

Figure 5.

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PSY1 and PSY2 have core structures that are conserved in the members of class 1 isoprenoid biosynthetic enzymes. A, Alignment of protein sequences from six class 1 isoprenoid biosynthetic enzymes. PSY1 and PSY2 are from tomato. The 3V66, 4HD1, 5IYS, and 3AE0 represent Protein Data Bank (PDB) ID (https://www.rcsb.org/) of human squalene synthase, Alicyclobacillus acidocaldarius squalene synthase, Enterococcus hirae dehydrosqualene synthase, and Staphylococcus aureus dehydrosqualene synthase, respectively, with known experimental crystal structures. The red line indicates the conserved domain of hydrophobic flap, and the two yellow lines mark the conserved Asp-rich motif 1 and 2. The numbers show residue sites in the respective proteins. The black, pink, and blue shading represents the homology levels at 100%, ≥75%, and ≥50%, respectively. B, Tomato PSY 3D structures are similar to SQS and CrtM. The structures of human SQS (3V66) and E. hirae CrtM (5IYS) were generated from the experimental data online, and those of PSY1 and PSY2 were predicted. Colored patches correspond to the domains marked by the colored lines in (A). Three residues important for phytoene synthase activity identified in this study are marked in white. The top and bottom panels show the 3D structures as cartoons and surfaces, respectively.

The conserved feature in all class 1 isoprenoid biosynthetic enzymes is an α-helical core around a central active site channel in which one end is more hydrophilic, with two Asp-rich motifs for substrate binding, and the other end is predominantly hydrophobic with a flap connecting two helices (Pandit et al., 2000; Gao et al., 2012). As for SQS and CrtM, the predicted PSY162-412 or PSY286-438 structure contained a similar central active site channel (Fig. 5B). One end of the channel had two conserved Asp-containing sequence motifs, and the other end had a conserved flap (YAKTF; Fig. 5B). Both PSY1 and PSY2 also had a wall formed by the side chain of Phe residue (Phe-135PSY1 /Phe-159PSY2) in the conserved flap. Crystal structure analysis of the SQS protein suggests that this wall is a crucial structure of the central active site cavity (Pandit et al., 2000). Interestingly, it was noted that Phe-159PSY2 was adjacent to Tyr-160PSY2, the critical residue for high PSY2 activity.

To experimentally prove the importance of the flap residue Phe-159PSY2 for PSY2 activity, SDM was performed to generate F159I, F159Y, and F159N mutants of PSY2. The residue substitution with another aromatic residue F159Y was found to give a good level of lycopene production as PSY2, whereas the substitutions of F159I and F159N with other groups of amino acids dramatically reduced cell hue and lycopene levels (Fig. 6A). Examination of PSY abundance and GGPS and CrtI expression found no association between their expression levels and lycopene content (Fig. 6A; Supplemental Fig. S3B), indicating that the altered lycopene production was due to PSY activity differences. These results show a crucial role of Phe-159PSY2 or an aromatic residue in this site of the hydrophobic flap for PSY2 activity.

Figure 6.

Figure 6.

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Neighboring aromatic–aromatic combination is critical for PSY activity. A, SDM analysis of the impact of the Phe residue in the conserved flap for PSY activity. Top, Typical pellet color of E.coli cells harboring PSY2 and mutated PSY2. Middle, Immunoblot analysis of PSY protein levels in the E. coli cells and Ponceau S staining showing protein loading. Bottom, Relative lycopene level in the cells expressing PSY2 or mutated PSY2. Values are mean ± sd of three biological repeats. *P < 0.05 and **P < 0.01 indicate significant difference based on the Student–Newman–Keuls multiple range test. B, SDM analysis of the effect of the neighboring residue on PSY activity. Top, Typical pellet color of E. coli cells harboring PSY1 and mutated PSY1. Middle, Immunoblot analysis of PSY protein levels in the E. coli cells. Bottom, Relative lycopene level in the cells expressing PSY1 or mutated PSY1. Values are mean ± sd of three biological repeats. *P < 0.05 and **P < 0.01 indicate significant difference. C, Interatomic interactions around the residues Phe-30 and Tyr-31 of CrtM (PDB ID: 5IYS) with ligand FPS revealed by Arpeggio (Jubb et al., 2017). The interatomic interactions include VdW interactions, polar contacts, hydrogen bonds, aromatic contacts, and hydrophobic contacts. Ligand FPS is marked in red. The noncovalent interactions are illustrated by the blue dashed bonds. Amino acids are shown in green. D, Location of Phe-135PSY1/Phe-159PSY2 and its neighboring residue (Asn-136PSY1/Tyr-160PSY2) in PSY1 and PSY2 3D structures. Top and bottom show the 3D structures as cartoons and surfaces, respectively.

Both PSY2 and PSY1 contained a Phe residue (Phe-135PSY1/Phe-159PSY2) in the conserved flap. Noticeably, we found that the PSY1(N136Y) mutant greatly enhanced lycopene production to a level similar to PSY2 (Fig. 4C; Supplemental Fig. S2), suggesting an aromatic neighboring residue being important for PSY activity. To test this hypothesis, various additional mutants of PSY1 at Asn-136 site were generated via SDM. Like PSY1(N136Y), substitutions of Asn with aromatic amino acids, namely, PSY1(N136F) and PSY1(N136W), were found to promote 3- and 2-fold, respectively, increases of lycopene production (Fig. 6B; Supplemental Fig. S5). By contrast, N136I, N136K, N136L, and N136T substitutions significantly reduced PSY1 carotenogenic activity (Fig. 6B; Supplemental Fig. S5). The altered lycopene levels were not associated with PSY protein level changes and GGPS and CrtI expression in the cells of these mutants (Fig. 6B, Supplemental Fig. S3C). These results demonstrate the critical importance of the neighboring aromatic–aromatic pair for PSY activity.

The neighboring residues Phe-159 and Tyr-160 of PSY2 correspond to CrtM Phe-30 and Tyr-31 (Fig. 5A). To see how this neighboring aromatic–aromatic combination influenced PSY activity, we utilized the web server Arpeggio (Jubb et al., 2017) to calculate and visualize the interatomic interactions of Phe-30 and Tyr-31 with ligand S-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl] trihydrogen thiodiphosphate (FPS) based on the experimental crystal data of CrtM (PDB ID: 5IYS). Abundant interatomic interactions between Phe-30 and FPS were observed, but no interatomic interactions between Tyr-31 and FPS were found (Fig. 6C), indicating that only the Phe in the conserved hydrophobic flap was involved in ligand binding. PSY 3D structures also supported this because the side chain of Phe-135PSY1/Phe-159PSY2 in the conserved flap was exposed to the central active site channel, and the residue Asn-136PSY1/Tyr-159PSY2 was on the surface of PSY proteins (Fig. 6D).

The aromatic side chain conformation associated with the channel is known to be crucial for protein function (Kayikci et al., 2018). To see whether variants of the neighboring residue affected the orientation of the Phe side chain, we investigated the experimental crystal data of homologous proteins (5IYS, 4HD1, 3V66) and Bacillus subtilis SQS 3WE9 (Summers et al., 1987). All have the central channel structure and the conserved Phe wall, but the different neighboring residues resulted in diverse side chain orientations of Phe (Supplemental Fig. S6).

Evolutionary Features of the Key Residues Associated with PSY Activity

Two residue site variations (Asn-136PSY1/Tyr-160PSY2 and Gly-198PSY1/Ala-222PSY2) were found to affect PSY activities (Fig. 4). A neighboring aromatic–aromatic residue combination (Phe-159PSY2 with Tyr-160PSY2) endowed tomato PSY2 with strong carotenogenic activity. To identify the evolutionary features of PSY proteins at these crucial sites for PSY1 and PSY2 activity, we examined the orthologs of PSY1 and PSY2 in 360 tomato accessions (Lin et al., 2014; Supplemental Table S1). These accessions include wild relatives of tomato, cherry tomatoes (Solanum lycopersicum var. cerasiforme), and cultivated tomatoes (Lin et al., 2014). The large-scale sequence alignment analysis showed that all accessions had Asn and Gly in the corresponding residue sites Asn-136PSY1 and Gly-198PSY1 for PSY1 and Tyr and Ala in the corresponding residue sites Tyr-160PSY2 and Ala-222PSY2 for PSY2. The alignment analysis indicates that these two variation sites are highly conserved for PSY1 and PSY2 in tomato varieties. In addition, Phe in the hydrophobic flap is conserved in all these tomato accessions.

We also investigated the orthologs of PSY in the relative species of tomato including potato, tobacco (Nicotiana tabacum), pepper (Capsicum annuum), and goji (Lycium barbarum). The same residue variations at these two corresponding residue sites were found only in potato PSY1 and PSY2 (Fig. 7A), suggesting that the weak PSY1 appeared before the tomato and potato genomes diverged. Moreover, we checked these two residue sites in the putative orthologs of PSY1 and PSY2 from 213 plant and algae uniproteins from UniProtKB (https://www.uniprot.org/; Supplemental Table S2). Only two additional PSY proteins contained different substitutions at the corresponding residue site Asn-136PSY1/Tyr-160PSY2, which are from Crocus sativus and Gentiana lutea (Fig. 7A; Supplemental Table S2). At the Gly-198PSY1/Ala-222PSY2 position, only one additional PSY protein from Selaginella moellendorffii contained the same residue Gly as PSY1 (Fig. 7A; Supplemental Table S2). All PSYs from other plant species shared the same two amino acids as PSY2 in the corresponding residue sites of Tyr-160PSY2 and Ala-222PSY2.

Figure 7.

Figure 7.

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Evolutionary features of the key residues associated with PSY activity. A, Alignment of partial protein sequences around the key residues for tomato PSY activity divergence. Protein sequences are from plant uniproteins in UniProtKB (https://www.uniprot.org/). Black and pink shading represents homology levels at 100% and ≥75%, respectively. Black triangles indicate numbered sites. B, Sequence logo of the aromatic–aromatic core structure around Phe wall in organisms of land plants, algae, cyanobacteria, fungi, and bacteria. The bigger sizes of the residue letters represent the higher frequency of occurrence in UniProtKB (https://www.uniprot.org/). Red residue letters show higher frequency, and black residue letters show occasional occurrence. C, Carotenogenesis of PSY from tomato, daffodil (Narcissus pseudonarcissus), and maize (Zea mays). Typical pellet color (top) and relative lycopene levels (bottom) of the E. coli cells harboring various PSYs are shown. D, Alignment of partial protein sequences including the targeted residues associated with daffodil PSY activity. Protein sequences are from plant uniproteins in UniProtKB. Black and pink shading represents homology levels at 100%, and ≥75%, respectively. The red triangle indicates the numbered site. E, Effects of NpPSY and mutated NpPSY on promoting lycopene production. Typical pellet color (top) and relative lycopene levels (bottom) of the E. coli cells harboring NpPSY and mutated NpPSY are shown. F, SDM analysis of PSY2. Left, The relative lycopene level in the cells expressing PSY2 or mutated PSY2. Right, Three-dimensional structure shows the location of the mutated residues. Green lines indicate those mutated amino acids. Values are mean ± sd of three biological repeats. *P < 0.05 and **P < 0.01 indicates significant difference based on the Student–Newman–Keuls multiple range test.

In addition, the aromatic–aromatic core structure (Phe-159PSY2 and Tyr-160PSY2) was also investigated in more organisms that included plants, algae, cyanobacteria, fungi, and bacteria (Supplemental Table S2). The “Phe wall” residue at the corresponding Phe-159PSY2 site was highly conserved in almost all of the organisms, and only the Tyr residue occasionally appeared in several bacteria species (Fig. 7B). The neighboring Tyr residue was also highly conserved in algae and cyanobacteria, whereas some different residues such as Phe and Ser appeared frequently in fungi (Fig. 7C). In bacteria, the residue showed diversity, but Tyr was still the most common residue following with Phe, His, and Ala (Fig. 7C).

Both daffodil PSY (NpPSY) and maize PSY (ZmPSY) contain Tyr and Ala at the corresponding postions of 160 and 222 of tomato PSY2 (Fig. 7A). However, NpPSY is regarded as a weak enzyme and ZmPSY1 as a strong enzyme in promoting carotenogenesis, as demonstrated in Golden Rice 2 (Paine et al., 2005). We also introduced these two PSY genes into the bacterial system and confirmed higher activity of ZmPSY1 than NpPSY in promoting lycopene synthesis (Fig. 7C). Tomato PSY2 showed higher activity for lycopene production than ZmPSY1 (Fig. 7C). To test whether there were residue sites that were negatively associated with NpPSY enzyme activity, two residue sites His-175NpPSY and Phe-204 NpPSY were selected for SDM analysis. This analysis was based on variations of conserved sites among the putative orthologs from 213 plant and algae uniproteins (UniProtKB; Fig. 7D; Supplemental Table S2). The NpPSY(H175P) mutant caused about 2-fold enhanced NpPSY activity for lycopene production, whereas NpPSY(F204L) did not change lycopene level in the BL21 bacterial cells (Fig. 7E). His-175NpPSY is in close proximity to the first Asp-containing motif 169DELVD173 (Supplemental Fig. S7), indicating that the position is important for NpPSY activity.

The specialized enzymes that catalyze a single reaction on a particular substrate have been evolving toward high activities, although most are mediocre (Newton et al., 2018). To further investigate the evolutionary selection of some residues at or around active sites of PSY, four additional residue sites (Thr-163, Met-166, Ile-177, and Val-179) of tomato PSY2 were selected for SDM analysis. They corresponded to the key residues at or around active sites in determining substrate specificity (Umeno et al., 2002) and binding (Pandit et al., 2000). These mutants all significantly reduced lycopene production (Fig. 7F), indicating optimization of these residues during PSY enzyme evolution. However, the mutated form PSY2(A200D), the corresponding residue in improving cassava PSY activity (Welsch et al., 2010), was able to further promote PSY2 activity with approximately 1.5-fold more lycopene production (Fig. 7F).

Strong Transcription of PSY1 Compensates for Its Weak Enzyme Activity during Tomato Fruit Ripening

Mature tomato fruit accumulate massive amounts of lycopene (McQuinn et al., 2018; Yazdani et al., 2019). However, PSY1 as a responsible enzyme was less effective in promoting carotenogenesis (Figs. 2 and 3). To explain this conundrum, we investigated the transcription pattern of PSY1 in tomato fruit using the online RNA-sequencing data (Sato et al., 2012). PSY1 showed very strong transcription when tomato fruit became mature: about a 180-fold increase from 20 to 3900 reads per kilobase per million mapped reads, which represents the seventh most abundant transcript among all tomato genes in mature fruit (Fig. 8A). In addition, based on the fruitENCODE data (http://www.epigenome.cuhk.edu.hk/encode.html), we compared the transcription patterns of the fruit-specific PSY1 during fruit development and ripening in five species (i.e. tomato, peach [Prunus persica], watermelon [Citrullis vulgaris], melon [Cucumis melo], and papaya [Carica papaya]) that all overproduce carotenoids in mature fruits (Lü et al., 2018). The fruit-specific PSY1 genes all showed a substantial increase in transcription during fruit ripening, but a greater increase was found only in tomato (Fig. 8B). The strong transcription of PSY1 might have evolved to compensate for its weak enzyme activity during tomato fruit ripening to achieve high levels of carotenoid biosynthesis.

Figure 8.

Figure 8.

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Transcription of fruit-specific PSY. A. RNA-sequencing data from Sato et al. (2012) show strong PSY1 expression when tomato fruit become mature. The PSY1 transcript is indicated in red. Transcript abundance was quantified as reads per kilobase per million mapped reads (RPKM). B. FruitENCODE data (Lü et al., 2018) show that tomato PSY1 expression (as RPKM) has a distinctive increase among various ripe fruits during fruit development and at mature stages. Inset, PSY expression in watermelon, melon, and papaya on a different scale. The fruit developmental stages represent 7, 17, 27, 37, 42, and 47 DPA for tomato; 63 (immature) and 98 DPA (mature) for peach; 10, 18, 26, and 34 DPA for watermelon; 10, 20, 30, and 40 DPA for melon; and 30, 90, 120, and 150 DPA for papaya.

DISCUSSION

Phytoene synthase is a major rate-limiting enzyme of carotenogenesis and comprises a small family with up to three members in plants. Although the tissue-specific expression of PSY paralogs has been well researched in plants (Li et al., 2008; Qin et al., 2011; Fantini et al., 2013; Stauder et al., 2018; Ahrazem et al., 2019), limited information is available on their respective activities in catalyzing carotenoid biosynthesis and on the amino acid residues critical for their activity divergence. In this study, we found that tomato PSY1 and PSY2 had different abilities in promoting carotenogenesis and discovered the key residues responsible for their activity divergence. These key residues were highly conserved among tomato accessions and plant species. Because PSY activity contributes greatly to the pool size of carotenoids in plants (Paine et al., 2005), the insights obtained and tools used in this study will benefit the application of synthetic biology to rationally design PSY for targeted metabolic engineering and breeding of carotenoid-enriched crops.

Tomato Fruit-Specific PSY1 Is Less Effective in Promoting Carotenogenesis

A most noticeable characteristic of ripening in tomato fruit is a dramatic increase of carotenoids (Fraser et al., 1994; Ronen et al., 1999). Because PSY1 controls fruit carotenoid levels (Fraser et al., 1999; Kachanovsky et al., 2012), it is reasonable to assume that this fruit-specific PSY1 has strong enzymatic activity for the massive carotenoid biosynthesis in ripening tomato fruit. By using both plant and bacterial systems to examine the individual effects of PSY1 and PSY2 on carotenoid accumulation, we found that tomato PSY1 had lower activity than PSY2 even though mature PSY1 and PSY2 share 86% high sequence identity. PSY1 showed activity similar to NpPSY (Fig. 7A), consistent with the results reported in a maize callus system (Paine et al., 2005). A previous study describes that the tomato chromoplast-specific PSY1 has less affinity for its substrate GGPP (Fraser et al., 2000). Although ripe tomato fruit contain the highest carotenoid levels, PSY in green fruit has higher enzymatic activity (Fraser et al., 1994). These studies also indicate potentially lower activity of PSY1 than PSY2. It appears that the green tissue–specific PSY possesses high catalytic activity, possibly due to the adaption of active photosynthesis requirements.

Why did tomato select for or retain weak PSY for its massive carotenoid accumulation in fruit? Tomato PSY1 is a PSY2 paralog derived through a gene-duplication event (Sato et al., 2012). After duplication, the two genes are maintained in the genome, likely owing to subfunctionalization from evolutionary selection. Tomato fruit ripening is achieved with transcriptional up-regulation of ripening-related genes including those of carotenogenesis (Lü et al., 2018). Over 180-fold increase of PSY1 from young to mature fruit was noticed during tomato fruit ripening (Sato et al., 2012). This weak PSY1 might better adapt to the highly upregulated transcription of PSY1 in mature fruit. In another aspect, the upregulation pattern of PSY1 was not observed during nonred fruit ripening of wild tomato species such as Solanum pennellii (https://solgenomics.net/organism/Solanum_lycopersicum/genome). It was speculated that recruitment of active carotenoid metabolism for lycopene happened later in evolution (Giorio et al., 2008). Therefore, the transcriptional activation machinery of PSY1, along with other carotenogenic genes in red tomato fruit, appeared after weak PSY1 formation. A weak PSY1 might also be related to its function in tomato flowers, as PSY1 expresses in both fruit and flowers. It is possible that weak PSY1 activity was evolutionally selected or retained for the adaption to chromoplast development. Notably, PSYs from tomato, daffodil, and pepper, known to enable high carotenoid biosynthesis in chromoplasts, are all fairly ineffective in promoting carotenogenesis (Paine et al., 2005).

Aromatic–Aromatic Association for High PSY Activity

Protein 3D structures are valuable to gain insights into protein function and to pinpoint the residue sites important for enzyme activity. Although there are no experimentally determined structures for PSYs, the advanced and matured protein structure homology modeling technique (Waterhouse et al., 2018) allowed us to generate and investigate 3D models of the PSY proteins based on human SQS and bacterial CrtM structures (Pandit et al., 2000; Lin et al., 2010). Plant PSY is an enzyme related to SQS and CrtM that catalyze squalene and dehydrosqualene synthesis, respectively. The predicted 3D core structures of tomato PSY showed a highly conserved structure similar to other isoprenoid biosynthesis C1 superfamily members (Pandit et al., 2000; Lin et al., 2010). Congruently, a single residue mutation gives CrtM a specific activity to accept GGPP as a substrate like PSY (Umeno et al., 2002). The SQS inhibitor squalestatin that binds to the SQS active sites was also found to inhibit PSY activity (Fraser et al., 2000).

Interestingly, the crucial residue (Asn-136PSY1/Try160PSY2) associated with the PSY1 and PSY2 activity variation neighbored a Phe (Phe-135PSY1/Phe-159PSY2) in a conserved two helices–connecting hydrophobic flap (YAKTF; Fig. 5A). The Phe side chain in the flap is known to form a wall of a hydrophobic cavity. The wall is proposed to be crucial in the central active site channel, yet no experimental data were available to support its importance (Pandit et al., 2000). By using SDM analysis, we provided evidence to support the critical role of an aromatic residue at this flap site for high PSY activity (Fig. 6A). An aromatic residue likely necessitates the forming of the wall of a hydrophobic cavity in the active site (Pandit et al., 2000). Moreover, we demonstrated the critical importance of an aromatic–aromatic combination in the flap site for PSY activity (Fig. 6A; Supplemental Fig. S5). It is likely that this neighboring aromatic–aromatic combination is needed to maintain the side chain conformation of the Phe wall for high PSY activity. Such an aromatic–aromatic association in the flap site could imply the requirement with other members of the isoprenoid biosynthesis C1 superfamily for high activities.

Selection in the Evolution of PSY Activity

Although PSY1 from 360 tomato accessions along with potato has Asn in the corresponding amino acid (Asn-136PSY1), this residue site of PSY in other plants, algae, cyanobacteria, many fungi, and some bacteria is Tyr as in PSY2. The neighboring Phe in the hydrophobic flap is extremely highly conserved and maintained by natural selection in almost all of the organisms (Fig. 7B). Mutations of these two neighboring amino acids into nonaromatic residues dramatically reduced PSY activity, showing conservation of the key residues for high activity. Similarly, alterations of some other residues in or around PSY core structures also significantly decreased PSY activity (Fig. 7F). These findings suggest that the key residues of PSY active sites are optimized under selective pressure during enzyme evolution toward high activity. This is consistent with the general belief that specialist enzymes evolved to become highly efficient (Nam et al., 2012). However, the evolutionary processes with gene duplication, divergence, and allelic variations also gave rise to PSY with catalytic activities different within and among species. PSY like most enzymes is far from a “perfect” catalyst during enzyme evolution (Newton et al., 2018), which offers the potential to be engineered toward higher enzyme activity.

Plasticity of PSY and its Implications for Carotenoid Enrichment in Crops

PSY activity contributes greatly to the pool size of carotenoids synthesized in plants (Giuliano et al., 2008; Sun et al., 2018). PSY is plastic with respect to its sequence variation and enzyme activity. Indeed, PSY sequence divergence, even at a single amino acid level, has been linked with carotenoid content change in species such as cassava, banana, and wheat (Welsch et al., 2010; Crawford et al., 2011; Mlalazi et al., 2012). As demonstrated in this study, the Phe-135PSY1/Phe-159PSY2 residue in the conserved hydrophobic flap and its neighboring Asn-136PSY1/Tyr-160PSY2 residue strongly affected PSY activities (Fig. 6).

Although the evolutionarily highly conserved key residues might be optimized toward high enzyme activity, clearly some additional sites outside of the key motifs or active sites are also important for PSY activity. A change of residue 191 from Ala to Asp in cassava PSY has been shown to alter carotenoid production in both the bacterial system and transgenic cassava (Welsch et al., 2010). Mutagenesis of the corresponding cassava residue in both tomato PSY1 and PSY2 also resulted in greatly increased PSY activity for lycopene production (Figs. 4D and 7F). Alteration of NpPSY(H175P), a residue in close proximity to the first Asp-containing motif of NpPSY, doubled lycopene synthesis (Fig. 7E). In maize PSY1, variations at Asn-168 and Thr-257, with the latter causing remote structural changes in the loop region in close proximity to Asp-rich motif 1, alter PSY1 plastid localization and morphology (Shumskaya et al., 2012).

Although the codon usage bias in bacteria sometimes might prevent direct comparison of PSY activities among various plant species, the engineered bacterial system used here provides an excellent tool for rapidly identifying the residues affecting carotenogenic activity. In many cases, it has been demonstrated that the increased carotenoid synthesis in bacteria is consistent with enhanced carotenoid production in the plant system, as shown in this study and in other reports (Welsch et al., 2010; Shumskaya et al., 2012; Camagna et al., 2019; Nogueira et al., 2019). Studies of the effects of carotenogenic enzyme sequence variations on carotenoid biosynthesis will have far-reaching implications and open up the potential to create new functional variants for targeted metabolic engineering and breeding of nutritionally enriched crops.

MATERIALS AND METHODS

Plant Materials and Growth

Arabidopsis (Arabidopsis thaliana; Columbia-0) and Nicotiana benthamiana were grown in soil under long-day condition with 14 h of light and 10 h of dark in a computer-controlled growth chamber at 23°C. Arabidopsis calli were induced following the protocol as described by Yuan et al. (2015a). The surface-sterilized Arabidopsis seeds were sown directly on plates containing callus induction medium. Following cold stratification for 2 d, the plates were placed in a growth incubator with 16 h of light and 8 h of dark at 23°C for 5 d and then in the dark for 2 weeks to induce callus growth and color development. NFZ treatment of 3-week-old leaves of Arabidopsis PSY transgenic lines was done as reported (Welsch et al., 2018). Callus and leaf samples were collected, frozen immediately in liquid nitrogen, and stored at –80°C.

Plasmid Construction and Plant Transformation

To generate tomato (Solanum lycopersicum) PSY1 and PSY2 overexpression constructs, PSY1 and PSY2 were amplified using gene-specific primers (Supplemental Table S3) from complementary DNA (cDNA) of tomato red fruit and young leaves, respectively, and cloned into pCAMBIA1300S with a 35S promoter (Zhou et al., 2011) to generate 35S:PSY1 and 35S:PSY2 constructs. For subcellular localization analysis, PSY1 and PSY2 open reading frames (ORFs) without stop codons were cloned and introduced into the pGPTVII vector to produce 35S:PSY1-GFP and 35S:PSY2-GFP constructs.

To generate stable PSY1 and PSY2 transgenic lines, 35S:PSY1 and 35S:PSY2 constructs were introduced into Agrobacterium tumefaciens GV3101 and transformed into Arabidopsis by floral dipping. Independent homologous T3 transgenic lines were selected based on seedling segregation on the selection medium. For 35S:PSY-GFP transient expression in N. benthamiana, leaves of 3-week-old plants were infiltrated with A. tumefaciens GV3101 containing 35S:PSY1-GFP or 35S:PSY2-GFP plasmid and checked for GFP fluorescence 2 d after infiltration, as described by Zhou et al. (2015).

RNA Extraction and RT-qPCR

Total RNA from 100 mg of Arabidopsis calli or 2 mL of Escherichia coli cells (optical density [OD] = 1.0) was isolated using Trizol reagent, following the manufacturer’s instruction (Life Technologies). The cDNA was synthesized with the PrimeScript cDNA Synthesis Kit (TaKaRa). Real-time RT-qPCR was carried out with SYBR Green Master Mix (Bio-Rad) on a CFX384 Touch Realtime PCR Detection System (Bio-Rad) using gene-specific primers (Supplemental Table S3). The expression levels of carotenogenic genes in Arabidopsis calli were normalized to the Arabidopsis actin gene. The expression of the CrtI gene in bacteria was normalized to the E. coli 16S gene.

Protein Extraction and Immunoblot Analysis

Total proteins from Arabidopsis calli and bacterial cells were extracted using a phenol method, as described previously by Yang et al. (2007). Protein concentrations were determined using the Bio-Rad protein assay kit. Equal amounts of proteins from different samples were separated by 10% (w/v) SDS-PAGE gel and blotted onto polyvinylidene difluoride membrane (EMD Millipore). The membrane was first stained with Ponceau S for protein loading and incubated with anti-PSY or anti-GGPS antibody for immunoblot analysis, as described in Lu et al. (2006), Zhou et al. (2015), and Welsch et al. (2018). The protein signals were detected using the enhanced chemiluminescence reagents (GE Healthcare).

Carotenoid Extraction and Analysis

Carotenoids from Arabidopsis calli and NFZ-treated leaves were extracted using the method described in Lopez et al. (2008). Briefly, 200 mg of tissues were ground in 1 mL 80% (v/v) acetone, mixed with 0.5 mL of ethyl acetate, and partitioned with 0.4 mL of water. Following centrifugation at 12,000g for 10 min, the upper carotenoid-containing phase was evaporated to dryness. The dried samples were dissolved in 200 μL of ethyl acetate before analysis.

To analyze the extracted carotenoids from plant tissues, a Waters Acquity UPC2 system with an Acquity UPC2 HSS C18 SB column (1.8 μm, 3.0 × 100 mm) was used (Yazdani et al., 2019). Carotenoids were separated using a gradient with CO2 and methanol in a total of 6 min. The individual carotenoids were identified based on their retention times and their characteristic absorption spectra and quantified using a β-carotene calibration curve (Li et al., 2012). At least three biological replicates were analyzed for each sample.

To analyze lycopene levels in E. coli cells, lycopene from the bacterial cells was extracted with 300 μL of acetone three times, and the supernatants were measured at an absorbance of 474 nm using spectrophotometry, as described by Camagna et al. (2019). The lycopene amounts were expressed as levels relative to each other.

Engineered Bacterial System for Lycopene Production

E. coli bacteria do not produce carotenoids. To generate a bacterial system that synthesizes lycopene, the Arabidopsis GGPS11 ORF without its putative transit peptide sequence was cloned into the vector pACYC to form pACYC-GGPS plasmid. A bacterial carotene desaturase (CrtI) from Pantoea ananatis was inserted into the first multiple cloning site of pCDF to generate pCDF-CrtI plasmid (Camagna et al., 2019). The ORFs of PSY variants were amplified using gene-specific primers (Supplemental Table S3) and cloned individually into the second multiple cloning site in the vector pCDF containing CrtI. The plasmids of pACYC-AtGGPS11 and pCDF-CrtI-PSY were transformed into E. coli BL21 competent cells. Overnight culture from a single colony was subcultured into 5 mL of fresh selective medium containing spectinomycin and chloramphenicol antibiotic and grown at 37°C to OD600 0.5 before inducing protein expression by adding 0.5 mm isopropyl-β-d-thiogalactopyranoside. The culture was incubated at 28°C for 4 h more, and its OD600 was measured. The cells were spun down and used. At least three colonies from each transformation were inoculated for analysis.

SDM of PSY

To specifically alter an amino acid of a PSY protein, the PSY gene was cloned into the pCDF-CrtI vector. The Agilent QuikChange Primer Design program (http://www.genomics.agilent.com/primerDesignProgram.jsp) was used to design SDM primers (Supplemental Table S3). The QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) was utilized to mutagenize PSY genes following the manufacturer’s instruction.

Structural Modeling and Sequence Analysis of PSY

For the predictions of PSY protein structures, an automated protein structure homology-modeling server SWISS-MODEL (https://www.swissmodel.expasy.org/) was used to search the template that optically matched the sequences of tomato mature PSY proteins. A dehydrosqualene synthetase (CrtM) template (PDB ID: 5IYS) was selected and used via the comparative modeling methods to generate the PSY protein models (Waterhouse et al., 2018). Interatomic interactions around the residues Phe-30 and Tyr-31 of CrtM (PDB ID: 5IYS) with ligand FPS were analyzed using Arpeggio (Jubb et al., 2017). Figures were prepared using PyMol (http://www.pymol.org).

Alignments of protein sequences and the corresponding residues as well as homology tree analysis were carried out using the DNAman software (Lynnon BioSoft, Vaudreuil). Conserved domain analysis was performed using a Conserved Domains search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). PolyPhen-2 analysis (http://genetics.bwh.harvard.edu/pph2/) was used to predict the tolerance in proteins (Adzhubei et al., 2010). Sequence analysis of PSY1 and PSY2 in 360 tomato accessions (Lin et al., 2014) and uniproteins in UniProtKB (https://www.uniprot.org/) was performed using BLAST (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/). The sequence logo at the aromatic–aromatic core structure of PSY was generated based on Weblogo3.1 (http://weblogo.threeplusone.com) using the sequence data from Supplemental Table S2.

Statistical Analyses

The SAS statistical software was used to compare the statistical difference based on the Student–Newman–Keuls multiple range test at significance levels of P < 0.05 (*) and P < 0.01 (**).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: PSY1 (M84744), PSY2 (EF534738), NpPSY (X78814), and ZePSY (U32636); PSY1 (P08196), PSY2 (A9Q2P8), SQS (P37268), and CrtM (I6T9U8).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We are grateful to Dr. Jie Ye (Huazhong Agricultural University) for providing the sequences of PSY1 and PSY2 in 360 tomato accessions. We thank Tara Fish (U.S. Department of Agriculture-Agricultural Research Service) and Meghna Prasad (Cornell University) for their technical assistance.

Footnotes

1

This work was supported by the Agriculture and Food Research Initiative competitive award grant no. 2016-67013-24612 from the U.S. Department of Agriculture National Institute of Food and Agriculture, the U.S. Department of Agriculture-Agricultural Research Service base fund, the International Postdoctoral Exchange Fellowship Program of China Postdoctoral Council (no. 20160024 to H.C.), and the HarvestPlus research consortium (grant no. 2014H6320.FRE to R.W.).

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