In recent years, the industry of Actinidia arguta has sprung up, and its fruit has become more and more popular in the market. At present, it has been widely cultivated in Liaoning, Zhejiang, Henan and other regions, and has become a new cultivated species of Actinidia Lindl plants with rapid development (Li, 2017, Graduate School of Chinese Academy of Agriculture Sciences, pp.14). Actinidia arguta is rich in resources, and its fruit is rich in flavonoid anthocyanins and antioxidants, which makes the fruit show a variety of colors (He and Giusti, 2010). Flavonoid anthocyanins play an important role in the anthocyanin synthesis pathway. As the main catalytic enzyme in the downstream region of the flavonoid synthesis pathway, colorless leucoanthocyanidin dioxygenase (LDOX) relies on 2-ketoglutarate ion and Fe²⁺ to catalyze the oxidation of colorless anthocyanins, thereby transforming colorless anthocyanins into colored anthocyanins (Appelhagen et al., 2011; Chung et al., 2010; Xie et al., 2004). The LDOX gene was cloned and identified from maize (Zea mays L.) A2 mutant according to transposon labeling technology (Menssen et al., 1990). At present, the LDOX gene has been successively isolated from Arabidopsis thaliana (Xie et al., 2004), Chrysanthemum × morifolium cultivar Argus (Chung et al., 2010), Fragaria × ananassa (Chen et al., 2018), Vitis vinifera (Li et al., 2016), Musa nana (Che et al., 2020). In addition, the study of LDOX in Arabidopsis thaliana showed that the seed coat color became lighter due to the reduction of anthocyanins and proanthocyanidins of LDOX allele mutants (Bowerman et al., 2010). The study found that the color difference of kiwifruit pulp color development into red, green and yellow is related to the initial color conversion process of red pulp involved in LDOX gene (Zhang et al., 2015). In grape research, it was found that LDOX gene in the peel of red varieties is higher than that of white varieties (Wen, 2005, College of Food Science and Technology, China Agricultural University, pp.4-5). The above research showed that as an enzyme at the end of the anthocyanin synthesis pathway, LDOX gene plays a key role in the accumulation of anthocyanins.

LDOX not only plays an important role in the coloring of Actinidia arguta, but also plays an important role in improving plant stress resistance (Zhang, 2016). At present, although we have a certain understanding of the structure and function of LDOX gene, we have not deeply analyzed the details of LDOX gene. Therefore, based on the transcriptome (RNA-seq) data of Actinidia arguta in NCBI, we searched the cDNA sequence of LDOX gene of Actinidia arguta, by using the method of bioinformatics, we can further explore the physical and chemical properties, protein structure characteristics, homology and functional annotation of LDOX in Actinidia arguta, which lays a theoretical foundation for further exploring the functional mechanism of LDOX in Actinidia arguta.

1 Results and Analysis

1.1 Homology and phylogenetic analysis of LDOX gene sequence in Actinidia arguta

The nucleotide and amino acid sequences of LDOX gene of 29 species were downloaded from the online database of NCBI nucleotide blast and protein BLAST applications. The nucleotide and amino acid sequences of Actinidia arguta were compared and analyzed by MegAlign (ClustalW) in DNAStar Lasergene application. It was found that the nucleotide and amino acid sequences of LDOX gene of Actinidia arguta were similar to those of Camellia Sinensis (79.2%, 87.6%), Vaccinium Ashei (79.3%, 85.1%), Durio zibethinus (76.6%, 85.0%), Morus notabilis (76.60%, 83.1%), Eucalyptus grandis (75.50%, 81.7%), with 75%~80% or higher. It has low similarity with Elaeis guineensis (70.0%, 69.6%) and Chrysanthemum × morifolium cultivar Argus (68.6%, 77.3%).

The phylogenetic tree of LDOX of 28 species was constructed by using the Neighbor Joining (NJ) method in MEGA 7.0 after ClustalW comparison, with an average genetic distance of 0.294. In conclusion, the sequence can be used for phylogenetic analysis (Figure 1). Actinidia arguta is closely related to Camellia sinensis, Vaccinium corymbosum, Vaccinium ashei, Lonicera japonica, Daucus carota and Nicotiana sylvestris, which gathered into one branch. Ten conserved motifs were identified by MEME software. The conserved motifs of the LDOX protein of Actinidia arguta were analyzed, and the motifs were named Motif1, Motif2, Motif3, Motif4, Motif5, Motif6, Motif7, Motif8, Motif9 and Motif10 (Table 1). Actinidia arguta and other species except Garcinia mangostana, Vigna angularis and Vigna radiata var. radiata contained the same conserved motif at the same amino acid position, which showed that LDOX protein is highly conservative in various species.

Figure 1 The LDOX proteins phylogenetic tree and the distribution of the conservative motifs Note: The left side was the phylogenetic tree of LDOX protein; the color box on the right represented the conservative base sequence distribution of the gene

Table 1 The protein conserved motif of LDOX Note: The ordinate indicated the conservative nature of amino acids, and the height of amino acid letters indicated the frequency of occurrence; The abscissa represented the position of the amino acid in the sequence

1.2 The cis acting element of LDOX gene promoter

In order to further explore the main potential functions of LDOX, this study used the neural network promoter prediction and Plantcare online server to predict the cis response elements of LDOX gene in Actinidia arguta (Table 2), and further analyzed the type, number and location of LDOX promoter elements. The results showed that the functional elements were contained in transcription factors, mainly including CAATbox (55 bp), ABRE (719 bp), ARE (169 bp), AuxRR-core (279 bp), CGTCA-motif (704 bp), G-box (719 bp), LTR (172 bp and 502 bp) and TGA-element (348 bp). Among them, ABRE responds to drought response in ABA signaling pathway; ARE can play an important role in anaerobic-induced regulatory element; AuxRR-core and TGA-element are important regulatory factors conducive to the development of endosperm, cotyledon and lateral roots. In addition, many regulatory elements of light stimulus response have also been identified in this study. Therefore, it was judged that LDOX is an essential element in photosynthesis and flowering regulation of light responsive plants.

Table 2 The cis element analysis of LDOX gene promoter

1.3 Analysis of LDOX gene protein characteristics

Using Protparam server to analyze the physical and chemical properties of the LDOX protein of Actinidia arguta, it can be seen that LDOX encodes 355 amino acids, with a relative molecular weight of 39 964.73 u, a theoretical isoelectric point of 5.61, and the number of alkaline being larger than that of acidic range. To sum up, this protein is an alkaline amino acid; The total average hydrophilic gravity is negative, so it is speculated that it is a hydrophilic protein with relatively high thermal stability. The total number of positive and negative charge residues is Asp + Glu=53 (positive), Arg+Lys=41 (positive), the molecular formula is C₁₇₈₉H₂₈₁₃N₄₇₇O₅₃₂S₁₄, the total number of atoms is 5 625, the instability index is 53.02, the fat coefficient is 87.07, and the average hydrophilicity is -0.390. The highest content of LDOX is Glu (11.5%), followed by Leu (9.9%) and Gly (7.0%), and the lowest content is Trp (1.4%) and Cys (1.4%) (Figure 2A).

Figure 2 Analysis of physical and chemical properties of LDOX protein Note: A: Amino acid compositions of protein encoded by the LDOX; B: Hydrophilicity /hydrophobicity analysis of LDOX

ProtScale online server predicted and analyzed that the strongest hydrophilic site of LDOX was located at position 64 (-3.511) of glutamic acid Glu (E), while methionine Met (M) was the strongest hydrophobic site (2.167), which was located at position 190 (Figure 2B). The hydrophilic region of LDOX protein was larger than the hydrophobic region, which further verified the prediction results of the total average hydrophilic protein that LDOX protein was a hydrophilic protein. Moreover, it can be seen (Figure 3) that no signal peptide region and transmembrane structure were found in LDOX protein.

Figure 3 Analysis of physical and chemical properties of LDOX protein Note: A: LDOX protein signal peptide prediction; B: Prediction of transmembrane structure of LDOX protein; C value: The value of the original shear site; S value: The value of signal peptide; Y value: The value of the integrated shear site

1.4 Phosphorylation and glycosylation sites of LDOX protein

Protein phosphorylation regulates its activity and function. NetPhos 3.0 Server was used to predict the phosphorylation modification sites of the LDOX protein of Actinidia arguta. The prediction results were shown in Figure 4. The LDOX protein sequence of Actinidia arguta contains 13 Ser phosphorylation sites (12, 16, 60, 109, 130, 159, 166, 170, 173, 174, 191, 239, 269), 12 Thr phosphorylation sites (4, 8, 52, 78, 164, 172, 184, 236, 242, 262, 323, 349) and 6 Tyr phosphorylation sites (25, 116, 145, 168, 175, 286).

Figure 4 Prediction of phosphorylation sites of LDOX

NetOGlyc 3.1 Server predicted that there were two O-glycosylation sites in LDOX protein, Thr⁴ and Thr³²³ respectively. The N-glycosylation site Asn¹⁰⁷ was predicted by NetNGlyc 1.0 Server that it exists at the LDOX residues (107~109) of Actinidia arguta. And it was predicted that Thr⁴ exists in both phosphorylation and glycosylation modification, but its glycosylation score (0.539) is less than the phosphorylation score (0.799), so it is considered that it is more likely to be phosphorylated.

1.5 Domain and advanced structure prediction of LDOX protein

The LDOX protein was analyzed and predicted by the “Conserved Domains” tool in NCBI. It was found that the LDOX protein of Actinidia arguta includes the 2-ketoglutarate-Fe²⁺-dioxygenase family structure (positions 48-347) (Figure 5).

Figure 5 LDOX protein conserved domain prediction

By analyzing and predicting the advanced structure of the LDOX protein in Actinidia arguta, it was found that the proportions of α-helix, extended-chain, β-turn and random coil were 34.65% (123), 16.06% (57), 6.20% (22) and 43.10% (153) respectively. In conclusion, the structure of LDOX protein mainly contains random coil, while α-helix and β-turn structure accounts were less (Figure 6).

Figure 6 The secondary structure prediction of LDOX Note: Blue: α-helix; Red: β-sheet; Green: β-turn; Purple: Random coil

The tertiary structure prediction model of SWISS-MODEL for LDOX protein of Actinidia arguta found that (Figure 7) the similarity between LDOX protein sequence of Actinidia arguta and 2brt.1.A template sequence in Blast database was as high as 78.96%. At the same time, the prediction results showed that QMEAN was 0.92, QMEAN was -0.26, and the coverage was 0.98%, indicating that the structural model was reasonable.

Figure 7 The tertiary structure prediction of LDOX protein

1.6 Interaction network construction and function enrichment analysis of LDOX enriched protein of Actinidia arguta

The database String was used to predict the functional relationship of LDOX protein in Actinidia arguta and the interconnected genes in the network diagram of protein interaction (Arabidopsis thaliana was selected as species mode). The results showed that there was a certain interaction relationship between LDOX and flavonol synthase (FLS) and other genes, and it was likely that they interacted with each other in completing a certain item or a certain physiological process (Figure 8). GO and KEGG enrichment analysis showed (Table 3) that LDOX interaction network genes participate in flavonoid biosynthesis and metabolism, mainly play a role in plant-type vacuolar membrane, and have the function of regulating catalytic activity and redox active molecules. KEGG enrichment function pathway is mainly flavonoid biosynthetic process and biosynthesis of secondary metabolites.

Figure 8 Function interaction network and co-expression of LDOX protein

Table 3 Functional enrichment analysis of LDOX genes

2 Discussion

Color plays an important role in the commodity value of fruits and is also one of the important indicators to evaluate the quality of fruits (Cui et al., 2020). Water soluble flavonoids mainly contain anthocyanins. The downstream synthetic gene LDOX gene plays an important role in the anthocyanin synthesis pathway. As an important regulatory hub in the regulation of color and metabolic genes, it is essential in the process of color appearance and change (Nakajima et al., 2001). This study used bioinformatics methods to predict and analyze the physical and chemical properties, structure and function of LDOX in Actinidia arguta, which can provide an important theoretical basis for further understanding the main functions of LDOX gene. Through the construction of phylogenetic tree, it was found that LDOX of Actinidia arguta is closely related to Camellia sinensis, Vaccinium corymbosum, Vaccinium ashei, Lonicera japonica and Daucus carota, indicating that LDOX is highly conservative in different species and conforms to the characteristics of biological evolution. No signal peptide region fragment was found in the sequence of Actinidia arguta. It was inferred that LDOX protein did not undergo protein transport. After initial synthesis on free ribosomes, it was retained in the cytoplasmic matrix as a catalyst to catalyze the formation of colorless anthocyanins to colored anthocyanins (Dong et al., 2010). Promoter prediction and results showed that a large number of acting elements were involved in the promoter sequence of LDOX gene of Actinidia arguta in response to different biological and abiotic stresses, indicating that the biological function of LDOX gene was related to stress induction, plant hormone induction and fruit maturation.

Branches of the flavonoid pathway in plants have a unified upstream pathway with the synthesis of proanthocyanidins and flavonols, which controls the biosynthesis of anthocyanins (Li, 2017, Graduate School of Chinese Academy of Agricultural Sciences, pp.14). All pathways are jointly participated by a series of catalytic enzymes. The first step of chalcone synthase catalyzed flavonoid pathway is that 4-coumarinyl-CoA and malonyl-CoA are catalyzed to synthesize chalcone. Then, chalcone isomerase catalyzes the synthesis of trihydroxyflavanone. Under the catalysis of F3H, DFR and other enzymes, trihydroxyflavanone synthesizes three colorless anthocyanins. Finally, Colorless anthocyanins form various anthocyanins under the catalytic modification of LDOX and UDP-flavonoid glycosyltransferases (UFGT). LDOX enzyme has not only sequence similarity with F3H and FLS, but also functional complementarity. Current studies have found that LDOX mainly replaces FLS enzyme catalytic activity in vitro. For example, Medicago Sativa (Pang et al., 2007), Oryza sativa (Xu et al., 2012) and Petunia hybrida (Nakajima et al., 2001) LDOX recombinant protein can replace FLS enzyme to catalyze diaminoflavonol to flavonol in vitro. LDOX protein also plays a bifunctional catalytic activity function in Reaumuria trigyna (Zhang, 2016, Inner Mongolia University, pp.23-26), but unlike other plant LDOX catalytic properties, LDOX recombinant protein cannot replace the catalytic function of FLS in vitro. Instead, it replaces F3H to catalyze the production of triamcinolone acetonide (Xu et al., 2012). In vivo, LDOX catalyzes the modification of the central carbon ring of flavonoids, which can catalyze the formation of procyanidins and anthocyanins from colorless anthocyanins (Welford et al., 2005). In addition, PAL protein, as an important hub of plant primary metabolism and phenylpropane secondary metabolism, plays a key role in regulating the synthesis of secondary metabolites such as flavonol, anthocyanin and lignin in plants. In this study, the interaction protein network of LDOX was constructed, and the enrichment analysis of GO and KEGG showed that the interaction between LDOX and F3H, UFGT, FLS as well as PAL, and the biosynthesis and metabolism of flavonoids were significant enrichment functional pathways. This indicates that LDOX, together with F3H, UFGT, FLS and PAL, plays a role in the regulation of Actinidia arguta color. The construction of LDOX protein network lays a solid foundation for further exploring the pathway and functional mechanism of LDOX involving in flavonoids.

3 Materials and Methods

3.1 Acquisition of LDOX gene sequence and construction of phylogenetic tree of different species

Take “Actinidia arguta leucoanthocyanidin dioxygenase (LDOX)+Actinidia arguta” as the keyword, and use NCBI (https://www.ncbi.nlm.nih.gov/) database to search and download the nucleotide and protein sequences of the LDOX gene of Actinidia arguta. The nucleotide (MK896863.1) and protein sequence (QGN03641.1) of the LDOX gene of Actinidia arguta were obtained, and the similarity was compared and analyzed by using the BLAST online server tool in NCBI. The nucleotide and protein sequences of the LDOX gene of species with high homology were downloaded one by one. The NJ method in MEGA7.0 was used to carry out the phylogenetic tree between Actinidia arguta and other species, and the Bootstrap value was set to 1 000.

3.2 Prediction and analysis of LDOX gene promoter in Actinidia arguta

Neural Network Promoter Predictio and PlantCARE online server were used to predict and analyze the transcription start sites and potential cis elements of the promoter of LDOX gene in Actinidia arguta.

3.3 Physicochemical properties and subcellular localization of LDOX gene in Actinidia arguta

The molecular formula, molecular weight, acidity and alkalinity, isoelectric point, stability and hydrophobicity of LDOX were predicted and analyzed by using ProtParam tool and ProtScale tool in ExPASy data analysis system. SignalP 4.0 server, TMHMM v. 2.0 server, NetPhos 3.1 server, NetNGlyc 1.0 server and NetOGlyc 3.1 server software were used to predict and analyze the signal peptide and transmembrane region of the LDOX in Actinidia arguta species, the serine, threonine or amino acid phosphorylation sites, and the O-glycosylation and N-glycosylation sites of LDOX in Actinidia arguta. The conserved region of LDOX protein was analyzed using MEME database.

3.4 Prediction of the advanced structure of LDOX protein in Actinidia arguta

The secondary structure prediction and analysis software SOPMA and the tertiary structure model prediction and analysis software SWISS-MODEL were used to predict and analyze the advanced structure of LDOX protein.

3.5 Prediction of LDOX protein interaction network and functional enrichment analysis of Actinidia arguta

STRING database was used to construct the interaction network of proteins closely related to LDOX. Arabidopsis thaliana was selected as a model species with a reliability value of 0.7, and the number of interacting proteins was within 10. Go and KEGG pathway enrichment analysis of different proteins in the predicted protein interaction network was carried out through the functional annotation software DAVID.

Authors’ contributions

LZP completed the experimental design, experimental research and the writing of the first draft of this study; LLY, SY, ZY and LDS participated in the completion of data and manuscript proofreading; YWZ was the designer and person in charge of the project, guiding the experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by “Xing-Liao Talents Plan” Project of Liaoning Province (NO.XLYC1902015) and Special Project of Guiding Local Scientific and Technological Development by the Central Government of Liaoning Province (NO.2019JH6/10200005).

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