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Panax ginseng CYP703: Comparison
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Please note this is a comparison between Version 2 by Catherine Yang and Version 1 by Johan Sukweenadhi.

Cytochrome P450 (CYP) catalyzes a wide variety of monooxygenation reactions in plant primary and secondary metabolisms. Land plants contain CYP703, belonging to the CYP71 clan, which catalyzes the biochemical pathway of fatty acid hydroxylation, especially in male reproductive tissues. Korean/Asian ginseng (Panax ginseng Meyer) has been regarded as one of important medicinal plant for a long time, however the molecular mechanism is less known on its development.

  • cytochrome P450
  • PgCYP703A4

1. Introduction

The cytochrome P450 (CYP) superfamily of enzymes, which catalyze diverse substrates through oxygenation and hydroxylation reactions, are found in all organisms [1]. Plant CYPs are involved in a variety of biochemical pathways that produce primary and secondary metabolites, which constitute one of the largest families of enzymes in higher plants [2]. The diversification of land plant CYP families emerges during flowering plant evolution and specializes plant species with unique reactions [3]. The identification of CYP genes aids in understanding the evolution of various groups of enzymes and their conserved and diversified functions.
The CYP703 gene family was found across the land plant taxa, suggesting that it encodes an essential function [4]. Land plants developed specialized cell layer to adapt to the environment, including cutin synthesis. As cutin cover in most plant organ of land plant, male reproductive organ of flowering plants is covered with a hydrophobic polymer barrier derived from fatty acids. The production of functional gametophytes leads to a successful proliferation in flowering plants [5]. Male reproduction is a complex and highly coordinated biological process that includes the development of the male reproductive organ: the stamen that contains the microspores and pollen. The study of male reproduction is important not only for increasing crop yield but for producing improved crops, such as superhybrid plants, through the use of male sterile lines [6].
The CYP703 family is involved in the in-chain hydroxylation of mid-chain fatty acids, which is essential for the biopolymers of pollen exine, the outer wall of a pollen grain [7[7][8],8], and cutin, one of the most abundant lipid polymers and an important adaptive trait of plants to their terrestrial environment [9]. A. thaliana CYP703A2 (AtCYP703A2) catalyzes the conversion of medium-chain saturated fatty acids to the corresponding monohydroxylated fatty acids, with a preferential hydroxylation of lauric acid at the C-7 position [8]. In comparison, Oryza sativa CYP703A3 (OsCYP703A3) can only catalyze in-chain hydroxylation of lauric acid (C12), preferentially at position 7 [7]. The A. thaliana mutant, cyp703a2, exhibits impaired pollen development and a partial male sterile phenotype due to the lack of an exine [8]. O. sativa CYP703A3 resulted in a complete male sterility with an abnormal anther epidermis, as well as defective pollen exine, indicating that the diversified function of CYP703A during evolution [7]. Both participated in a conserved pathway of in-chain hydroxylation of lauric acid that is required for male reproductive development. Some male-specific gene sequences that determine sex, where CYP703 was found, have been identified in the Phoenix tree which belongs to dioecious species. [10]. 

2. Identification of CYP703 Gene in P. ginseng

CYP families often have many paralogs, but the CYP703 family was reported to be a single-gene-member family [8]. Analysis of the P. ginseng genome scaffold and CDS revealed that P. ginseng contains two scaffold sequences with high similarity to CYP703. Among the two scaffolds, scaffold 1562 contained a full-length CDS of CYP703 (Pg_S1562.26), which contained a two-exons and one-intron structure (Figure 1), similar to the A. thaliana gene structure. On the contrary, two CDS sequences, S6323.1 and S6323.2, present in scaffold 6323 were partial sequences of CYP703. Therefore, we concluded that the P. ginseng genome encodes just one PgCYP703 member, whereas the others are nonfunctional genes. The recent genome duplications of the P. ginseng genome [22][11] might explain the presence of two genes that can duplicated, in which one of the gene has retained the original sequence and function, whereas the other became a pseudogene. Similarly, CYP703A was noted as a single functional sequence in the poplar genome (Populus trichocarpa) [8].
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Figure 1. Analysis of gene and promoter structure of PgCYP703A4 and its pseudogene. Genomic sequence of scaffolds containing PgCYP703A4 and similar sequences were identified from the P. ginseng genome database (http://ginsengdb.snu.ac.kr/ accessed on 20 January 2022). (A) PgCYP703A4 gene was confimed as Pg_S1562.26 CDS, which encoded on Scaffold 1562. The coding regions (orange and green boxes) are interrupted by 1009 base pair (bp) intron. The upstream 1000-bp region from the translation start site has four POLLEN1LELAT52 binding-predicted sites and four MYBCORE binding-predicted sites. (B) A similar sequence structure was identified from Pg_scaffold6323 encoding two CDSs, assumed to be pseudogenes. The transcript was separated into two partial CDS sequences (Pg_S6323.2 and Pg_S6323.1). Dashed line indicates closed sequences between two scaffolds.
A putative ORF sequence, which had a length of 1119 bp and encoded 372 amino acids (Figure 1), was verified by sequencing, and an NCBI-BLAST search displayed the conserved superfamily CYP. There are three functionally reported genes: two CYP703A members registered in the Plant P450 Database (http://erda.dk/public/vgrid/PlantP450/, accessed on 10 December 2021) (CYP703A1 from a Petunia hybrida [23][12] and AtCYP703A2 [8]) and O. sativa CYP703A3 [7]. We named the CYP703 gene identified in P. ginseng as PgCYP703A4.

3. Phenotype Analysis of PgCYP3A4 Overexpressing A. thaliana

Due to difficulties in obtaining transgenic regenerated P. ginseng plants, we generated PgCYP703A4 overexpressing transgenic Arabidopsis (PgCYP703A4ox) to examine its functional role in planta (Figure 42A–D). The stable incorporation of the PgCYP703A4 gene and its heteroexpression was confirmed via RT-PCR (Figure 42B). PgCYP703A4ox produced slightly taller plants compared with the wild type, but not significant (Figure 42A). Notably, the siliques increased in size by 20% compared with the wild type and PgCYP703A4ox siliuqes contain higher number of seeds than wile type significantly (Figure 42C,D).
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Figure 42. Phenotype analysis of PgCYP703A4 overexpressing A. thaliana. (A) Growth phenotype of four different PgCYP703A4 overexpression lines and wild type at 2 week- and 7 week- old. Scale bar indicates 5 cm. (B) Detection of PgCYP703A4 transcription in transgenic A. thaliana’s rosette leaves. At actin served as the control. (C,D) Silique size of PgCYP703A4 overexpression lines. Scale bar indicates 5 mm. Values indicate mean of 20 biological replicates ± SD. * p < 0.05.
A.thaliana CYP703A3 mutant was reported to show impaired pollen walls lacking a normal exine layer, which leads to partial male sterility [8]. To determine how PgCYP703A4 affects pollen wall formation, we observed the anthers and pollen phenotype by semi-thin cross-section and SEM. The anther, pollen, and pistil of PgCYP703A4ox appeared similar to the wild type, whereas CYP703A3 exhibited aborted pollen without outer elegant wall formation. Therefore, pollen viability and reproductive organ function was not altered by PgCYP703A4 gene overexpression.

4. Overexpression of the Panax ginseng CYP703 Alters Cutin Composition of Reproductive Tissues in Arabidopsis

Plants have evolved a variety of enzymes for the in-chain α-, β-, and ω-hydroxylation of fatty acids. Hydroxylated fatty acids are the biosynthetic intermediates of plant biopolymers, such as cutin and suberin, which make up the barriers from land plant stress situations. Thus, fatty acid metabolic enzymes are critical for plants; however, the role of CYP members in controlling the development of P. ginseng has not been well studied.  CYPs constitute the largest family of enzymes in plant metabolism and represent plant evolution in terms of plant metabolism in development and adaptation, such as signaling, defense, and polymerization of complex chemical substances [37][13]. Among the 11 land plant clans, the CYP71 clan represents more than half of all CYPs in higher plants; consequently, a wide diversity of functions makes them more difficult to predict their preferred substrates than other clans [37][13]. In addition to CYP703, CYP77 family members, AtCYP77A4 and AtCYP77A6, can in-chain-hydroxylate fatty acids to form precursors of cutin [9,38,39][9][14][15]. Looking at their phylogenetic relationship, CYP703 diversified prior to the emergence of CYP77s as spore protectors. CYP703 is an ancient gene family that is required for land plants, whereas CYP77 is required in only angiosperms [37][13]. However, both CYP703A and CYP77A function as in-chain hydroxylases, compared with most other enzymes that are end-chain (ɷ) hydroxylase [30][16]. In addition, a part of the CYP71 clan, the CYP78 subfamily genes exhibited fatty acid hydroxylation reactions, particularly for short chains [24][17]. CYP78A family members regulate reproductive organ development but are more related to female organs. A. thaliana gene CYP78A9 was reported to be involved in the control of carpel shape [40][18]. The overexpression of CYP78A9 results in large, seedless fruit, although the metabolites have not been discovered [40][18]. O. sativa gene OsCYP78A13 promotes seed growth by regulating the embryo and endosperm size, as well as spikelet hull development [41][19]. PaCYP78A9 regulates fruit size in Prunus avium, showing increases in silique and seed size in A. thaliana by hetero-overexpression [42][20]. It is clear that the CYP71 clan has a large diversity of functions, but only CYP703 and CYP78 families of this clan, have the conserved PERF consensus, and both subfamiles are involved in plant reproductive development. Further studies are required to identify its positive relationship with biological function. In P. ginseng, reproductive development and functional studies are scarce. We previously identified a functional ortholog of AtCYP704B1, termed PgCYP704B1 [15][21]. The CYP704B family, which belongs to the CYP86 clan, is involved in the ω-hydroxylation of long-chain fatty acids. Altered exine in the pollen wall was detected in mutant of A. thaliana cyp704B1 [46][22], Brassica napus CYP704B1 [47][23], and O. sativa CYP704B2 [36][24]. However, O. sativa CYP704B2 also had an undeveloped anther cuticle and sterile male phenotype [36][24]. It is similar to CYP703A, although it is in a separate clan. Similarly, CYP701A and CYP88A, which belong to the CYP71 and CYP85 clans, respectively, act sequentially in the same pathway as ent-kaurene oxidase and kaurenolic acid oxidase, respectively [48][25]. With the early evolution of CYPs, CYP703 and CYP704 could be involved in cutin biopolymer synthesis, particularly for pollen wall polymers, for in-chain and ɷ-hydroxylation, respectively. This study was limited by the difficulty of obtaining flowers from transgenic P. ginseng. However, further studies on P. ginseng development is required to develop hybrid and male sterile system for breeding. Taken together, PgCYP703A4, a member of CYP703A in the CYP71 clan, and PgCYP704B1 [22][11], in the CYP86 clan, are similarly expressed in the P. ginseng tapetum and meiotic cells, and overexpression in A. thaliana affects fatty acid metabolism in siliques. Previous studies on A. thaliana and O. sativa examined knockout mutants displaying partial or full male sterility [7,9,36,46][7][9][24][22] and therefore did not further investigate the phenotype regarding fruit development. This requires further investigation to determine the role of hydroxylated fatty acids in sporopollenin synthesis and the development of the silique cuticle.

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