Structural and functional features of phytoene synthase isoforms PSY1 and PSY2 in pepper Capsicum annuum L. cultivars

The fruits of various pepper cultivars are characterized by a different color, which is determined by the pigment ratio; carotenoids dominate in ripe fruits, while chlorophylls, in immature fruits. A key regulator of carotenoid biosynthesis is the phytoene synthase encoded by the PSY gene. The Capsicum annuum genome contains two isoforms of this enzyme, localized in leaf (PSY2) and fruit (PSY1) plastids. In this work, the complete PSY1 and PSY2 genes were identified in nine C. annuum cultivars, which differ in ripe fruit color. PSY1 and PSY2 sequence variability was 2.43 % (69 SNPs) and 1.21 % (36 SNPs). The most variable were PSY1 proteins of the cultivars ‘Maria’ (red-fruited) and ‘Sladkij shokolad’ (red-brown-fruited). All identified PSY1 and PSY2 homologs contained the phytoene synthase domain HH-IPPS and the transit peptide. In the PSY1 and PSY2 HH-IPPS domains, functionally significant sites were determined. For all accessions studied, the active sites (YAKTF and RAYV), aspartate-rich substrate-Mg2+-binding sites (DELVD and DVGED), and other functional residues were shown to be conserved. Transit peptides were more variable, and their similarity in the PSY1 and PSY2 proteins did not exceed 78.68 %. According to the biochemical data obtained, the largest amounts of chlorophylls and carotenoids across the cultivars studied were detected in immature and ripe fruits of the cv. ‘Sladkij shokolad’ and ‘Shokoladnyj’. Also, ripe fruits of the cv. ‘Nesozrevayuschij’ (green-fruited) were marked by significant chlorophyll content, but a minimum of carotenoids. The PSY1 and PSY2 expression patterns were determined in the fruit pericarp at three ripening stages in ‘Zheltyj buket’, ‘Sladkij shokolad’, ‘Karmin’ and ‘Nesozrevayuschij’, which have different ripe fruit colors: yellow, red-brown, dark red and green, respectively. In the leaves of the cultivars studied, PSY1 expression levels varied significantly. All cultivars were characterized by increased PSY1 transcription as the fruit ripened; the maximum transcription level was found in the ripe fruit of ‘Sladkij shokolad’, and the lowest, in ‘Nesozrevayuschij’. PSY2 transcripts were detected not only in the leaves and immature fruits, but also in ripe fruits. Assessment of a possible correlation of PSY1 and PSY2 transcription with carotenoid and chlorophyll content revealed a direct relationship between PSY1 expression level and carotenoid pigmentation during fruit ripening. It has been suggested that the absence of a typical pericarp pigmentation pattern in ‘Nesozrevayuschij’ may be associated with impaired chromoplast formation.

It is known that primates, including humans, do not synthesize carotenoids de novo, but are in dire need of them, since, for example, β-carotene and α-carotene are precursors of vitamin A. The antioxidant activity of the carotenoids found in carotenogenic fruits and vegetables helps to reduce the risk of various diseases, such as certain types of cancer, age-related eye pathologies and cardiovascular diseases (Howard et al., 2000;Story et al., 2010;Giuliano, 2017). Among vegetable crops, pepper, which fleshy fruits are enriched with various types of carotenoids, is one of the main sources of antioxidants in the human diet. In this regard, the obtaining of new pepper varieties is an important task of modern breeding (Berry et al., 2019;Sun, Li, 2020).
Pepper species have different antioxidant levels, and many breeding programs use the natural variation to identify the characteristics of "exotic" allelic diversity and donors of specific carotenoid spectra. However, at present, closely related accessions of the same species are predominantly used, which often do not have strong phenotypic differences, usually observed when using wild relatives (Berry et al., 2019).
Carotenoids together with chlorophylls and anthocyanins determine the color of pepper fruits. It should be noted that carotenoids are the dominant pigments in ripe pepper fruits, while chlorophylls (sometimes together with anthocyanins) are in immature, growing fruits. In C. annuum cultivars, fruit color depends on the ratio of pigments, as well as on the stage of ripening: from green, yellow, white or purple in unripe fruits (mature fruit stage, MF), to orange, red, dark red, brown and sometimes almost black -in ripe fruits (ripe fruit stage, RF) (Levy et al., 1995;Márkus et al., 1999;Ha et al., 2007). Usually, sweet peppers are harvested at the technical ripeness stage (blanche fruit, intermediate ripe stage, IR), and hot peppers at biological ripeness (RF). The pepper fruit ripening is accompanied by the transition of tissues containing chloroplasts to tissues containing chromoplasts. In chromoplasts, chlorophylls degrade, but the synthesis of carotenoids continues, which, unlike chlorophylls, are able to accumulate in specialized globular structures (Osorio, 2019). This leads to a decrease in the chlorophyll content, the accumulation of carotenoids and, as a consequence, to a change in the ripening fruit color.
Unlike tomato, in ripe fruits of which the main carotenoids are lycopene and β-carotene, in pepper fruits, carotenogenesis goes further -to the formation of xanthophylls; carotenoid spectrum in ripe pepper fruits is represented by major concentrations of red pigments -capsanthin and capsorubin, as well as by various combinations of minor amounts of orange and yellow pigments β-carotene, β-cryptoxanthin, lutein, zeaxanthin, anthraxanthin and violaxanthin (Giuffrida et al., 2013;Mohd Hassan et al., 2019).
Carotenoid pigments are isoprenoid molecules obtained as a result of successive transformations of the universal precursor, isopentenyl pyrophosphate. Several reactions convert this compound into geranylgeranyl pyrophosphate (GGPP), two molecules of which condense head-totail by phytoene synthase to form phytoene, the precursor of all carotenoids (Fraser et al., 2000).
Thus, phytoene synthase is a key regulator of carotenoid biosynthesis, supplying the main substrate -phytoene (Fraser et al., 2000). This enzyme is encoded by the PSY gene, the expression of which is influenced by intermediate and final products of the pathway (Welsch et al., 2003;Kacha-novsky et al., 2012;Enfissi et al., 2017). Several types of phytoene synthases have been identified in plants, and phytoene synthase activity depends on the type of enzyme and its intracellular location (Shumskaya et al., 2012). In Arabidopsis thaliana, only one PSY gene was identified (Zhou et al., 2015), while in tomato Solanum lycopersi cum, three, and the protein products of these genes have different localization: PSY1 -in fruit plastids, PSY2 -in leaf plastids, PSY3 -in root plastids (Stauder et al., 2018). In pepper C. annuum, two genes are currently known that encode phytoene synthases, one of them is mainly locali zed in the leaf plastids (PSY2), the other -in the fruit plastids (PSY1) (Thorup et al., 2000;Kilcrease et al., 2015). Accordingly, in tomato and pepper, PSY2 transcripts are mainly present in photosynthetic green tissues, while PSY1 is found in mature fruits of both crops and in tomato flower petals (Giorio et al., 2008;Kilcrease et al., 2015;Berry et al., 2019;Filyushin et al., 2020). However, both phytoene synthases can be transcribed in all plant organs (Stauder et al., 2018).
This study is focused on identifying the genes of phytoene synthases PSY1 and PSY2 in C. annuum cultivars, assessing their intervarietal variability, both structural and functional, as well as possible correlations between the expression of these genes and fruit pigmentation.

Materials and methods
Plant material. Individual plants of nine C. annuum cultivars were used in the research: eight cultivars of sweet pepper (Nesozrevayuschij, Karmin, Shokoladnyj, Sladkij shokolad, Ratunda, Maria, Gogoshary, and Zheltyj buket) and one hot pepper cultivar (Mechta hozyayki) ( Table 1). The plants were grown in a greenhouse at the Federal Scientific Vegetable Center (FSVC, Moscow Region).
Comparative structural analysis of the PSY1 and PSY2. Alignment and analysis of the obtained nucleotide and amino acid sequences were performed using the MEGA 7.0 (https://www.megasoftware.net/). Known sequences of C. annuum PSY1 (Gene ID: 107868281) and PSY2 (Gene ID: 107859651) were used for comparative analysis. Conserved domains in encoded proteins were determined using the NCBI-CDD (http://www.ncbi.nlm. nih.gov/Structure/cdd/wrpsb.cgi) and UniProtKB (https:// www.uniprot.org/). The functional significance of each amino acid (aa) residue substitution was predicted using the PROVEAN (http://provean.jcvi.org/index.php). By radical substitutions is meant those substitutions that can presumably affect the folding of the protein or its functionality.
PSY1 and PSY2 expression pattern in fruits of the analyzed pepper cultivars during ripening. Total RNA was isolated (RNeasy Plant Mini Kit, QIAGEN, Germany) from fruit pericarp at three developmental stages (MF, IR, and RF). The resulting preparations were purified from DNA impurities (RNase free DNasy set, QIAGEN, Germany), evaluated qualitatively and quantitatively (spectrophotometrically and by electrophoresis in 1.5 % agarose gel), and used for the cDNA synthesis (GoScript™ Reverse Transcription System, Promega, USA).
The sum of chlorophylls and the sum of carotenoids in fruit pericarp (together the skin and pulp) were determined spectrophotometrically in chloroform-methanol extracts; the pigment content was calculated using the formulas (Lichtenthaler et al., 1987;Solovchenko et al., 2001), in two biological and three technical replicates.

Results and discussion
Characteristics of the PSY1 and PSY2 gene sequences and proteins encoded by them Previously, it has been shown that the PSY1 and PSY2 variability may determine the color of the pepper fruit (Cao et al., 2019;Filyushin et al., 2020). There fore, for this study, nine C. annuum cultivars were selected, which differ in fruit color during ripening: Nesozrevayu schij, Zheltyj buket, Shkoladnyj, Sladkij shokolad, Karmin, Ratunda, Maria, Gogoshary and Mechta hozyayki (see Table 1). Unripe fruit color of all analyzed cultivars was green or dark green, however, the dynamics of color change as they ripen differed among cultivars. In the cv. Nesozrevayuschij, the fruits remained green until biological ripeness, in the cv. Zheltyj buket they were yellow-green at the IR stage and yellow at the RF stage, in cv. Sladkij shokolad and Shkoladnyj, fruits were red-brown at both stages, while the other four cultivars had green-red fruits at the IR stage and red/dark red fruits at the RF stage (see Table 1).
For each of the nine pepper cultivars, the PSY1 and PSY2 gene sequences were determined, starting from the ATG codon (Table 2). The length of the PSY1 gene was 2844 bp in all analyzed cultivars. For comparison, C. an nuum cv. Zunla 1 PSY1 available in the NCBI database (Gene ID: 107868281) has the same size, while S. lyco persicum cv. Heinz 1706 (Gene ID: 543988) is longer (3302 bp). The length of the PSY2 gene in the studied cultivars was 2985 bp, with the exception of PSY2 from cv. Mechta hozyayki (2994 bp, due to the 9-nucleotide insert in the second intron) (see Table 2). The C. annuum cv. Zunla 1 PSY2 (Gene ID: 107859651) is also 2985 bp, whereas S. lycopersicum cv. Heinz 1706 PSY2 (Gene ID: 543964) is 3032 bp. The variability of the PSY1 and PSY2 genomic sequences in pepper accessions was 2.43 % (69 SNPs) and 1.21 % (36 SNPs), while 16 and 15 SNPs were localized in exons, respectively. Compared to S. lyco persicum cv. Heinz 1706 PSY1 and PSY2, PSY1 and PSY2 of the pepper cultivars contained 1072/128 and 818/100 (gene/exons) SNPs.
The coding part of the PSY1 and PSY2 genes consisted of six exons and in all studied cultivars was 1260 and 1299 bp, respectively (see Table 2). Found differences in cDNA length were due to the presence of insertions in exons I and VI of PSY2. Most of the identified SNPs were concentrated in exon III of PSY1 (7 SNPs, 43.75 % of all exon substitutions) and in exon VI of PSY2 (6 SNPs, 40.0 %). Exon II of both genes was invariable and the most conserved with respect to the S. lycopersicum cv. Heinz 1706 PSY genes. Exon I of both genes turned  out to be the most polymorphic in comparison with the S. lycopersicum PSY genes. Note that since in this work, a limited number of cultivars (nine) were analyzed, the data on gene polymorphism are applicable only to the set of analyzed cultivars. The PSY1 and PSY2 nucleotide sequences have been translated. The putative proteins PSY1 and PSY2 of all analyzed cultivars were 419 and 432 aa, respectively (see Table 2), contained a conserved phytoene synthase domain HH-IPPS (130-412 and 26-310 aa, according to UniProtKB, and 75-405 and 92-430 aa, according to NCBI-CDD) and the N-terminal transit peptide TP (1-129 and 1-25 aa, according to UniProtKB, and 1-74 and 1-91 aa, according to NCBI-CDD). TP cleavage sites in all possible cases were invariant within the analyzed set of cultivars.
Compared to the C. annuum cv. Zunla 1 and S. lycoper sicum cv. Heinz 1706 PSY1 and PSY2, in pepper cultivars, PSY1/PSY2 contained 9/15 and 46/43 aa substitutions, respectively. Out of nine substitutions in PSY1, seven were radical (r) and only two were neutral (n), while all r-substitutions were in the conserved domain, and two n-substitutions were in the transit peptide (Fig. 1). The nC59Y substitution was typical for PSY1 of almost the entire studied set of cultivars, except for the cv. Sladkij sho-kolad, and all radical substitutions were cultivar-specific. The most variable were PSY1 of cv. Maria and Sladkij shokolad (see Fig. 1).
In the PSY2, of 15 aa substitutions nine were radical. Phytoene synthases PSY2 of cv. Mechta hozyayki, Gogoshary, Ratunda, and Shokoladnyj did not differ from each other or contained nT430A, while each of the other cultivars had one or two r-substitutions in the HH-IPPS domain (see Fig. 1).
The presence of radical aa substitutions in PSY1 and PSY2 of the analyzed cultivars can affect the mature phytoene synthase folding, as well as enzyme ability to interact with protein partners and perform correct catalytic functions. Previously, it was shown that the PSY1 and PSY2 sequences are highly similar (Giorio et al., 2008;Cao et al., 2019). Comparison of the identified C. annuum PSY1 and PSY2 confirmed this observation.
Considering the UniProtKB data on the domain localization, HH-IPPS contains 21 variable sites specific for each of the PSY1 and PSY2 protein groups (see Fig. 1). Also, at the PSY1 domain C-terminus, two deletions, P422-S427del and L429del, were identified. The PSY1 and PSY2 domain sequence was highly conserved (92.86 %).
In contrast to the HH-IPPS domain, the TP sequence was found to be highly variable. In comparison with Structural and functional features of phytoene synthase isoforms PSY1 and PSY2 in pepper Capsicum annuum L. cultivars  14  71  59  72  186  117  210  178  214  180  288  191  320  236  321  290  389  309 383 387 393 394 395  PSY2 TP, PSY1 TP contained 29 aa substitutions (21.32 % of aligned length). Thus, the identity of TP in PSY1 and PSY2 in the studied pepper cultivars was 78.68 %; also, two insertions (insF31S33 and insG50) and four deletions (PSY2 num bering: N12del, D35del, L57-R62del, and S64-D65del) were identified in the PSY1 sequence. Apparently, diff erences in TP sequences may be responsible for the specificity of delivery of each of the phytoene synthases to different types of plastids, as was suggested earlier (Cao et al., 2019).
To confirm the structural similarity of the identified PSY1 and PSY2, cluster analysis was performed based on their genome-wide sequences in comparison with the known S. lycopersicum cv. Red Setter and C. annuum cv. Zunla 1 PSY1 and PSY2 (Fig. 2). On the dendrogram, the pepper cultivars were expectedly grouped into two large clusters combining the sequences PSY1 and PSY2, respectively (see Fig. 2). Within each cluster, C. annuum accessions formed a single closely related subcluster with insignificant internal bootstrap values (17-50) and the only reliable combination of cv. Maria and Zheltyj buket based on PSY1. The S. lycopersicum species occupied the base branch in each of the clusters.
Thus, the identified PSY1 and PSY2 of nine pepper cultivars, which differ in a ripe fruit color, were highly similar in structure, which suggests that they may preserve the conserved key functions of phytoene synthases in the carotenoid biosynthesis.

The content of chlorophylls and carotenoids in fruit pericarp during ripening
The total content of chlorophylls and carotenoids was measured in fruit pericarp during development in the analyzed pepper cultivars (see Table 1). It was shown that unripe fruit of all cultivars (stage MF) contains comparable amounts of chlorophylls and carotenoids, which characterizes the fruit tissues as photosynthetic. In IR fruits, chlorophyll content decreased by 1.46-5.60 times, depending on the cultivar. In ripe fruits (RF stage), chlorophyll was found in significant quantities in cv. Sladkij shokolad, Shokoladnyj and Nesozrevayuschij, and in small quantities in Gogoshary and cv. Mechta hozyayki. There were no chlorophylls in ripe fruits of cv. Zheltyj buket, Karmin and Maria.
In immature fruits, the carotenoid content was the highest in the cv. Shokoladnyj and Sladkij shokolad (48.5 and 61.0 µg/g), while in the other cultivars it ranged from 6.8 (Gogoshary) to 27.3 µg/g (Zheltyj buket) (see Table 1). In ripe fruits, the primacy remained with the cultivars forming chocolate-colored fruits: the highest carotenoid content was detected in the cv. Shokoladnyj (1009.90 μg/g), while in the cv. Sladkij shokolad, it was reduced by 1.7 times, and in the red-fruited cv. Karmin and Maria -by 1.94 and 2.48 times, respectively (see Table 1). Ripe fruits of the remaining four cultivars accumulated significantly less carotenoids. However, a similar low carotenoid content did not provide the similar ripe fruit color: Zheltyj buket -yellow, Mechta hozyayki -red, Nesozrevayuschij and Gogoshary -green and red-green, respectively. At the same time, cv. Nesozrevayuschij and Gogoshary fruits had on average two times less carotenoids in comparison with the cv. Zheltyj buket and Mechta hozyayki fruits (see Table 1).
In accordance with the obtained biochemical data, it can be assumed that red-fruited cultivars synthesize red pigments typical for peppers -carotenoids capsanthin and capsorubin. In brown-fruited cultivars, the color may be formed by two components -red carotenoids and green chlorophylls. The yellow or green color of fruits at the stage of biological ripeness is most likely determined by the presence of yellow-colored carotenoids (lutein, zeaxanthin) and chlorophylls, respectively.

PSY1 and PSY2 co-expression pattern in the fruit pericarp during ripening
Carotenoid accumulation in fruits is directly related to the PSY1 expression level (Meléndez-Martínez et al., 2010); however, although PSY2 is mainly expressed in photosynthetic tissues, its transcripts have also been found in fruits (Jang et al., 2020). In this study, PSY1 and PSY2 expression pattern was characterized in leaves and fruit pericarp (peel and pulp) at three ripening stages (MF, IR, RF) in four pepper cultivars (Fig. 3). The analysis included cv. Zheltyj buket, Sladkij shokolad, Karmin and Nesozrevayuschij, contrasting in the ripe fruit color (RF) -yellow, brown, dark red and green, respectively (see Table 1). Ripe fruits of cv. Karmin and Sladkij shokolad were characterized by a high carotenoid content (520.2 and 597.0 μg/g), while ripe fruits of cv. Nesozrevayuschij, and Zheltyj buket accumulated only 55.9 and 107.7 μg/g, respectively.
In the leaves of the analyzed pepper cultivars, the PSY1 expression levels varied significantly. For example, the levels were similar in cv. Nesozrevayuschij and Zheltyj buket (0.15 and 0.17, respectively), while three and ten times lower (0.054) in cv. Sladkij shokolad and Karmin (0.012), respectively (see Fig. 3). In the fruits of the analyzed pepper cultivars, a PSY1 expression pattern was similar -progressive upregulation during fruit ripening. At the final stage of ripening, in the ripe fruit, the maximum PSY1 expression level was observed in cv. Sladkij shokolad, and the minimum -in cv. Nesozrevayuschij (see Fig. 3).
Phytoene synthase PSY2 is considered to be more specific for photosynthetic tissues (Giorio et al., 2008). In green unripe fruit (stage MF), all analyzed cultivars were characterized by the presence of chlorophylls -the highest in cv. Sladkij shokolad and the lowest in cv. Nesozrevayuschij. Surprisingly, only in these two cultivars, ripe fruits also contained chlorophyll (in the former it was 3 times higher than in the latter).
In the leaves, the PSY2 expression level in cv. Sladkij shokolad was 2-3 times higher than in the other three cultivars, in which the expression was comparable (see Fig. 3). Besides, PSY2 transcripts were detected in the fruit pericarp at all ripening stages in all analyzed cultivars. In the immature fruit pericarp (stage MF), PSY2 expression was approximately at the same level in cv. Nesozrevayuschij (0.057), Sladkij shokolad (0.050), and Karmin (0.046), while in cv. Zheltyj buket, it was twice lower (0.025) (see Fig. 3). In all analyzed cultivars, the PSY2 expression level decreased as fruits ripen. In ripe fruits (stage RF), the PSY2 level was similar in cv. Nesozrevayushchij, Zheltyj buket, and Karmin, and 2.3-3.5 times higher in cv. Sladkij shokolad (see Fig. 3).
It can be assumed that the presence of PSY2 expression in pepper ripe fruits is associated with the chloroplast preservation. This was evidenced by the chlorophyll presence in the ripe fruit pericarp, for example, in cv. Nesozrevayushchij and Sladkij shokolad (see Table 1). However, cv. Zheltyj buket and Karmin also showed PSY2 ex pression in ripe fruits, while no chlorophyll was found there. Thus, it can be assumed that PSY2 can function not only in chloroplasts, but also in chromoplasts. Earlier, using pepper cv. MicroPep Yellow as an example (with lack of PSY1 gene transcription), it was shown that the synthe sis and accumulation of yellow pigments in fruit chromoplasts is associated with PSY2 expression (Jang et al., 2020).
The regulation of carotenoid biosynthesis and accumulation in pepper fruits is a complex process (Deruère et al., 1994;Kilcrease et al., 2015). In ripe fruits, carotenoids accumulate in chromoplasts in specialized globules, and if their formation is impaired, then carotenoids can be synthesized, but not accumulated (Osorio, 2019). Globule formation is controlled by the Orange protein, which at the same time prevents carotenoid degradation and stabilizes the phytoene synthase PSY activity (Osorio, 2019). Peel of cv. Nesozrevayuschij fruits, as they ripen, retained the green color, while the pulp color changes from light green to yellow-green. In accordance with this and in contrast to the other three analyzed cultivars, there was no significant increase in the carotenoid content (see Table 1), and the PSY1 transcription level in the ripe fruit of cv. Nesozrevayu schij was 1.6 and 2.1 times lower than that of cv. Karmin and cv. Zheltyj buket, respectively. It can be assumed that in cv. Nesozrevayuschij, a number of processes characteristic of fruit ripening, such as degradation of chlorophyll, transformation of chloroplasts into chromoplasts, and/or de novo chromoplast synthesis, are disturbed (Kilcrease et al., 2015;Berry et al., 2019). However, the fruits of cv. Nesozrevayuschij ripen (the seeds are fully formed and viable), although there is no noticeable change in the pericarp color. This confirms the previously shown lack of a relationship between fruit carotenogenesis and ripening (Fraser et al., 2007).

Conclusions
Thus, in the present study, in nine C. annuum cultivars, differing in ripe fruit color, PSY1 and PSY2 genes encoding phytoene synthases were identified and characterized; the co-expression pattern of these genes in the vegetative and reproductive organs, as well as possible relationships of the expression level with the total carotenoid content were determined. A direct correlation was found between the PSY1 gene expression level and carotenoid pigmentation of the fruit during ripening. It was shown that in the cv. Nesozrevayuschij, the pericarp pigmentation pattern, typical for pepper fruits during ripening, is disturbed, which may be associated with blocks in the chromoplast formation.