extfieldsofstudy: Chemistry Physics

text:
Characteristics of cheese analogue from corn extract added by papain and pineapple extract

Corn extract is an alternative ingredient which can substitute cow’s milk in making cheese analogue. The addition of pineapple and papain extract aims to produce cheese analogue with the physical and chemical desired. This study aims to 1) find out the optimum formula of additional papain and pineapple extract to produce extract corn-substituted cheese analogue, 2) Knowing the sensory, physical and chemical characteristics of cheese from corn milk produced from the best formula. The experimental design in this study was determined by Response Surface Methodology (RSM) using Central Composite Design with 2 factors studied, which are the amounts of pineapple extract (31.72-88.28 ml) and papain extract (89.64-150 mg). Variables observed include yield, moisture content, soluble protein, soluble solid and sensory variables. The optimum formula for producing cheese analogue substituted by corn extract is the addition of 40 ml pineapple extract and 130.82 mg papain extract. The yield of the formula is 19.2%. The cheese analogue has 5.908% of dissolved protein content, 12°Brix of dissolved solids and 24.3% of moisture content. The sensory characteristics (scale 1-5) of cheese analogue are whitish yellow (3.44), typical corn aroma (3.5), good taste (3.5), easy to spread (4.28) and preferred (3.5)

Introduction
Spread cheese is usually made by the coagulation of cow's milk or the other milk, for example is goat's milk. The lifestyle of today's consumers who want a balanced consumption of plant and animal foods raises innovation in cheese making by replacing a part or all of cow's milk with vegetable ingredients [1].
The cheese is called as cheese analogue. Cheese analogue, also called as cheese imitation, is defined as a cheese-like product, made from ingredients such as milk fat, vegetable fat, protein with the addition of emulsifiers, and also made through heat and mechanical energy [2].
Sweet corn extract is an alternative ingredient which can substitute the cow's milk in making cheese analogue. The type of cheese analogue which is widely made and used is spreadable cheese analogue because its manufacture is relatively easy and simple [3].
The coagulation process is an important stage which determines the texture and flavour of the cheese [4]. In order to help the coagulation process, rennet enzyme is added as the coagulant [5]. The rennet enzyme used as the coagulant of cheese is expensive; therefore it needs an alternative of rennet enzyme. According to [6] protease enzyme from plants can be used as an alternative in cheese making. One of the enzymes that can be used as the substitute The use of papain in cheese making can produce high yields and the characteristics of its cheese are similar to cheese made using rennet enzymes [7].
Cheese making requires an acidulant which aims to reduce pH. The decrease of pH is important in order to reach the isoelectric point which plays a role in the formation of the cheese texture. Pineapple has a pH of 2.3 and the presence of bromelin enzyme in pineapple can help the coagulation process in cheese making [8], [9]. According to [10] the addition of pineapple extract can coagulate milk in the production of soft cheese.
The objectives of this study are: 1) To find out the optimum formula for adding papain and pineapple extract to produce corn-substituted cheese analogue, 2) Knowing the sensory, physical and chemical characteristics of cheese from corn milk produced from the best formula.

Ingredients
The ingredients used in this study are sweet corn and pineapple bought from Wage Market, Purwokerto, fresh cow's milk (CV Tiga Putra), commercial papain (Paya(®)), Lactobacillus casei from the Biotechnology Research Center, Indonesian Institute of Sciences in Cibinong, West Java, Indonesia.

The making of cheese analogue
The cheese making includes the preparation of ingredients, corn steaming and corn seeds shelling. The next step is refining corn seeds by adding water (twice the weight of corn) to obtain the corn extract. The corn extract obtained was mixed with cow's milk which was then pasteurized for 30 minutes. The pasteurization was left to a temperature of 40 C and added with 1 ml of Lactobacillus casei liquid, 30g of sucrose, and 2Cl of CaCl 2 then incubated for 2 hours. The incubated milk was added by pineapple and papain extract. The curd formed is then separated by filter cloth and pressed, so that a semi-solid curd is produced. The curd was then cooked at 52 C for 15 seconds and added with 2g of NaCl and 4g of sucrose.

Experimental design
The experimental design in this study was determined by Response Surface Methodology (RSM) using (Central Composite Design) with 2 factors studied, which are the amount of pineapple and papain extract. The pineapple extract used was 31.72-88.28 ml; while the papain extract was used 89.64-150 mg. Based on the results of the randomization, 13 formulas were obtained (Table 1).

Initial product characteristics
The yield of cheese analogue produced was 8.32-9.28% ( Figure 1). This yield has a quadratic model with R-squared value of 0.35937 or not significant with the F value of 0.79 and p-value of 0.5912. This yield is almost the same as soft cheese from cow's milk using fruit extract which is 7.75 -10.29% [14]. The yield of this cheese analogue is lower than the cheese produced with acidification using fruit extract by [15] which states that cheese making using a fruit extract produces a yield of 14-18%. The cheese yield is also lower than the results [16] which states that the yield of cheese made using papain is 20.4-23.4%. According to [10], yield is affected by raw materials and composition, type of acidulant, as well as pasteurization and pressing method.
Increasing the amount of papain in cheese analogue making increases the yield of cheese. According to [17], the higher the protease enzyme added the more increase the yield. The more pineapple extract is added, does not affect the yield of cheese. This is different from [15] who states that the higher the concentration of fruit extracts, the lower the yield of  (Figure 2). The more addition of pineapple extract, the higher the dissolved solids. Pineapple extract contains the bromelin enzyme and is acidic so that it can reduce pH so that it reaches an isoelectric point. At the isoelectric point, milk clots occur which will separate whey and curd proteins. This will increase the dissolved solids in the cheese. The moisture content of this cheese analogue is 57.11% -76.87% (Figure 3). This is consistent with [15] that the moisture content of soft cheese without the ripening process has a moisture content of 52 -80%.  The addition of papain concentration does not significantly affect the moisture content. This is slightly different from [18] who states that the more addition of papain the more increase the moisture content of the cheese.
The more addition of pineapple to cheese making will reduce the moisture content of cheese.
The addition of acid to cheese making will accelerate the process of decreasing the pH. The lower pH the more acidic; so that the whey is easier to be separated from the curd. The ease of separating whey and curd results in low moisture content in the curd so that the moisture content of the resulting cheese is also low [18].
The cheese analogue produced has dissolved protein levels of 2.18-3.24% (Figure 4). The addition of papain increases the level of protein dissolved in cheese analogue. This is consistent with [16] that the higher the concentration of the papain enzyme is used, the higher the level of protein in traditional cheese produced. Papain is a type of protease enzyme, thus the more papain the more increase the proteolytic activity. Increasing the proteolytic activity will increase the breakdown of protein into peptides and amino acids so that the dissolved protein levels in cheese analogue increases [17]. This dissolved protein level is lower than the cheese analogue from cow's milk using papain, which is 12.14-22.56% [7]. This difference is caused by the differences in the materials used, because in this study the material used is cow's milk plus raw corn extract, where sweet corn has lower protein content than cow's milk. The increase in the amount of pineapple extract added does not significantly affect the level of cheese protein. This is slightly different from [19] that increasing the concentration of pineapple extract added will increase the protein content in cheese. This is because pineapple has a low pH which helps clump the protein so that in the process of separation, the level of protein in curd which is then processed into cheese is also high.
Topical cheese analogue making in this study did not use stabilizers. According to [19], the addition of stabilizers can increase dissolved protein level in cheese making.

The optimum formula and its verification
The optimization stage was done to get a formula with the most optimal response adjusted to the product characteristics desired. The most optimal response was obtained if the value of the desirability is close to 1. Weighting is done on each component to achieve the desired goal ( Table 2). The score of 1-5 shows that the higher the score, the more it is preferred to be optimized Based on the optimization with the criteria in Table 2, the optimum formula recommended is 40 ml of pineapple extract and 130.82 grams of commercial papain.
The formula can produce topical cheese analogue with the prediction set. The desirability value predicted from the optimum formula is 0.507. This value is actually very small, but this is the maximum result of the desirability that can be obtained in this study. The optimization result is then made the product and analysed to determine its accuracy, and the result can be seen in Table  3. The product verification produces data which does not match the prediction, but the result is better than the prediction. The yield of topical cheese produced was 19.2% greater than the predicted results (9.46%) ( Table 3). This yield is almost close to the results of [7] which makes cheese using papain and get cheese yields of 20.4-23.4%.
The optimized protein content was higher (5.91%) compared to the prediction (2.13%) ( Table 3). This dissolved protein level is lower than cheese analogue from the cow's milk using papain by [7] which is equal to 12.14-22.56%. This difference is caused not only by the raw material but also by the hydrolysis process using the bromelin enzyme in imperfect pineapple. According to [19], the low level of protein in cheese made using pineapple extract can be due to the low hydrolysis process. This results in wasted proteolysis and protein in whey. The soluble solid of the optimization result was higher (12 o Brix) compared to the prediction (3.06 o Brix) ( Table 3). The high total dissolved solid indicates the quality of good topical cheese. Total dissolved solid is the component of dissolved solids consisting of total sugars, pigments, organic acids and proteins [20].
The optimum colour cheese analogue formula has a value of 3.44 which is whitish yellow.
The yellow colour of the cheese analogue is thought to be due to the presence of beta-carotene from corn, but this article does not discuss beta carotene in cheese analogue. According to [21], sweet corn has beta carotene level of 400 SI.
The optimum aroma of cheese analogue the formula has a value of 3.5 which is typical of corn aroma. Aroma is affected by the components of volatile compounds present, which are dominated by the corn aroma.
The cheese analogue taste in this study was obtained from good optimum formula with a score of 3.5. The taste is dominated by the distinctive taste of corn and the panelists like the taste. This is in accordance with [21] who makes probiotic corn extract and increased corn extract solids can increase the consumer preference because of the typical corn flavour.
Ease of spreading is an important parameter in this study because the cheese analogue made is topical cheese analogue. The optimum cheese analogue formula has easy characteristics to be spread with a score of 4.28. This spread easiness characteristic is also supported by the results of testing, which is cheese that is easily attached to bread.
The favorite topical cheese produced from the overall optimum formula with a score (3.5), which is preferred. The assessment of the level of preference by panelists is a combination of the attributes (color, aroma, taste, topical power) which have the best value. This is the initial research of the development of cheese analogue substituted with corn extract. The yield of cheese analogue produced is still low so further research is needed to increase the yield, for example by adding fillers and stabilizers.

Conclusion
The optimum formula for producing the cheese analogue substituted with corn extract is the addition of 40 ml of pineapple extract and 130.82 mg of papain extract. The yield of the formula was 19.2%. The analog cheese has 5.908% of dissolved protein content, 12 o Brix of dissolved solids and 24.3% of moisture content. The sensory characteristic (scale 1-5) of cheese analogue is whitish yellow (3.44), typical corn aroma (3.5), good taste (3.5), easy to spread (4.28) and preferred (3,5).


extfieldsofstudy: Biology Medicine

s2fieldsofstudy:
Agricultural And Food Sciences
Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Genome-wide analysis of the WRKY gene family and their positive responses to phytoplasma invasion in Chinese jujube

Background The WRKY gene family is one of the most important families in higher plants. As transcription factors, they actively respond to biotic and abiotic stress and are also involved in plant development. Chinese jujube (Ziziphus jujuba Mill.) is the largest type of dried fruit tree in China in terms of production, but its production is largely limited by phytoplasma infection, and the information about the role of WRKY genes under phytoplasma stress was still limited. Results We identified 54 ZjWRKYs in the jujube genome and classified them into three subgroups according to conserved WRKY domains and zinc-finger structure. 41 ZjWRKYs were distributed on 11 of 12 pseudo chromosomes in Chinese jujube. The majority of ZjWRKYs were highly expressed in the seven examined tissues, indicating that they play multiple roles in these vegetative and reproductive organs. Transcriptome data showed that most of the characterised ZjWRKYs were highly expressed at later stages of fruit development. RT-qPCR demonstrated that the expression of 23 ZjWRKYs changed following phytoplasma infection, suggesting that they are involved in signalling pathways that respond to phytoplasma stress. Then, STRING analysis and yeast two-hybrid screening proved that some ZjWRKY proteins were interacting with ZjMAPKK proteins, which were also involved in phytoplasma invasion. Moreover, their differential expressions were further confirmed in resistant and susceptible jujube varieties under phytoplasma stress. These results suggest that ZjWRKYs play significant roles in phytoplasma tolerance and should be crucial candidate genes for jujube-phytoplasma interaction. Conclusions 54 ZjWRKYs in Chinese jujube were identified and classified into three subgroups. 41 ZjWRKYs were unevenly distributed along the chromosomes. The majority of ZjWRKYs were highly expressed in various tissues. Most of the ZjWRKYs were positive responses to phytoplasma invasion, and that provided candidate genes for the future studies of jujube-phytoplasma interaction. Electronic supplementary material The online version of this article (10.1186/s12864-019-5789-8) contains supplementary material, which is available to authorized users.

Background
The WRKY transcription factors (TFs) bind to a specific promoter sequence in the target gene, known as a W-box, and can positively or negatively regulate target gene expression. The WRKY proteins have one or two DNA binding domains that are 60 amino acids long and contain the conserved heptapeptide WRKYGQK followed by a zinc-finger motif C 2 H 2 (CX 4-5 CX 22-23 HXH) or C 2 HC (CX 7 CX 23-24 HXC) [1]. The WRKY family contains important transcription factors that have multiple functions in processes such as embryogenesis [2], trichome and seed development [3], leaf senescence [4], flowering [5], fruit and pollen development [6], biomass accumulation [7], secondary metabolite biosynthesis [8] and hormone signalling [9]. WRKY transcription factors are also crucial regulatory components of plant responses to pathogen infection. In Arabidopsis, several WRKY genes have been experimentally characterised and associated with responses to fungal or bacterial pathogens [10][11][12]. AtWRKY70 is required for R gene-mediated pathogen resistance, determining the balance between the SA-and JA-dependent defence systems [13,14]. Many WRKY genes also act in defence signalling; for example, AtWRKY38 and AtWRKY62 act as negative regulators of basal resistance towards bacterial pathogens [15]. In rice, overexpression of OsWRKY30 enhanced resistance to the rice sheath blight fungus Rhizoctonia solani and the blast fungus Magnaporthe grisea [16,17]. Owing to their important roles, the WRKY family has been widely studied in many plant species, such as Arabidopsis, rice, grape, apple, pear, and peach [18][19][20][21][22]. However, the information of this gene family in Chinese jujube and their roles under phytoplasma stress was still limited.
Chinese jujube is the largest type of dried fruit tree in China in terms of production [23] and the most important species of family Rhamnaceae. It is cultivated mainly for its fruits, which can be eaten fresh or dried or as raw materials for making Chinese herbal medicine. However, jujube production is threatened by several devastating diseases, such as jujube witches' broom disease (JWB). The genome of Chinese jujube was recently published [24,25], paving the way for further investigations. Our transcriptome data indicated that some WRKY genes respond to JWB phytoplasma infection. Since the WRKY family plays a crucial role in biotic stress response, identifying WRKY genes in Chinese jujube and determining their possible functions in response to phytoplasma stress have important significance.
Here, we report on the genome-wide analysis of the WRKY family in Chinese jujube. A non-redundant set of WRKY genes was identified in this species. Subsequently, chromosomal location was determined, phylogenetic and motif analyses were also performed as a base for further comparative genomics studies. Moreover, expression patterns of ZjWRKYs in various tissues and under phytoplasma stress were also investigated. The interacting proteins of ZjWRKYs were also screened. The ZjWRKYs involved in phytoplasma invasion were considered good candidates for subsequent studies of the jujube-phytoplasma interaction.

Identification of ZjWRKYs in Chinese jujube
A total of 54 non-redundant putative WRKY coding sequences (Table 1) were identified in the jujube genome sequence. The sequences were named from ZjWRKY1 to ZjWRKY54 according to their gene structure and motifs. The ORF length for ZjWRKY genes ranged from 522 bp (ZjWRKY26) to 2205 bp (ZjWRKY8), and they encoded proteins ranging from 173 to 734 amino acids (aa) in length, with predicted pIs ranging from 4.65 (ZjWRKY32) to 9.09 (ZjWRKY1) ( Table 1).
Previous genome evolution studies showed that Chinese jujube is closely related to species of the family Rosaceae [24,26], so the WRKY genes of three Rosaceae species (apple, pear and peach) and Arabidopsis were compared with that of Chinese jujube (Additional file 1). Compared with Arabidopsis, apple and pear [18,19,21], there are fewer WRKY genes in jujube, but the number was similar to that of peach [22]. The smaller number of WRKY genes in Chinese jujube and peach may be due to the occurrence of only one genome duplication event during the evolution of the two species [24,27]. Based on the above comparison, it was suggested that most of the expected WRKY genes in jujube were identified.

Conserved motifs and phylogenetic tree construction of ZjWRKYs
The phylogenetic tree of the ZjWRKY proteins was constructed by aligning multiple domain sequences (Fig. 1). The ZjWRKY proteins were classified into three groups (Group I, II and III) ( Table 1) according to their WRKY and zinc-finger motifs. The domain sequences in the ZjWRKY gene family were highly conserved. There were 8 motifs among ZjWRKYs and proteins in the same group had similar numbers and types of motifs (Fig. 2, Additional file 2). The WRKY domain (WRKYGQK, Motif 1) was highly conserved among the 54 proteins (Additional file 2) and only two of them contained variations. The group II proteins ZjWRKY25 and ZjWRKY26 showed a WRKY motif with one amino acid modifications (WRKYGKK) ( Table 1, Fig. 2). Motif 2 was also highly conserved except in the two Group III proteins ZjWRKY52 and ZjWRKY53. Motif 5 and Motif 8 were specific to groups I and III respectively.
Group I had 8 proteins (Table 1), that contained two WRKY motifs, and two C 2 H 2 zinc-finger motifs. Group II was the biggest group and included 34 proteins that contained a WRKY motif and a C 2 H 2 zinc-finger motif. According to the phylogenetic analysis, the 34 genes could be further divided into five subgroups (IIa to e) that included 3, 10, 11, 3 and 7 genes, respectively ( Table 1). The members of subgroups IIa, IIb, IId and IIe had a CX 5 CX 23 HX 1 H zinc-finger motif, while that of subgroup IIc had a CX 4 CX 23 HX 1 H structure (Table 1). Group III contained 12 proteins, and they had one WRKY motif and a C 2 HC zinc-finger motif (CX 7 CX 23 HX 1 C, Table 1).

The chromosomal location and gene structure of ZjWRKYs
Of the 54 ZjWRKY genes, 41 were mapped to 11 of 12 pseudo chromosomes in the jujube genome (Fig. 3), and 13 genes were located on 12 scaffolds (Table 1, Additional file 3). ZjWRKYs were not evenly distributed across the 11 pseudo chromosomes (Fig. 3). Ten ZjWRKYs (18.5%) were located on Chr. 11, whereas only one ZjWRKY gene was on Chr. 5 and 8 each. No ZjWRKY gene was found on Chr. 7. Additionally, the gene structure was highly conserved within each group, especially in groups IId, IIe, and III. We found that Group I genes contained more introns and were more complicated than genes in the other two groups (Fig. 4).

Expression profiles of ZjWRKYs in various tissues/organs
To investigate the tissue-specific expression of the jujube WRKY genes, RT-PCR was used to determine their expression patterns in seven tissues. The expression patterns of 26 ZjWRKY genes were analysed and are shown in Fig. 5a. Of the 26 ZjWRKY genes, six genes were actively expressed in at least five tissues, including ZjWRKY6, 9, 10, 13, 29, and 42. A total of 8 ZjWRKY genes (ZjWRKY2, 22, 25, 33, 36, 44, 45 and 48) were found to be upregulated in only one or two tissues, indicative of the tissue-specific expression of these genes.
ZjWRKY24 expression could only be detected in roots and old branches. The expression of the remaining genes was comparatively low in the different organs, suggesting that genes in the same group might have different functions. These results showed that most of the ZjWRKY genes had diverse tissue-specific expression patterns, indicating that ZjWRKYs play multiple roles in various organs. Moreover, a heat map of our RNA-Seq data highlighted differential expression of ZjWRKYs during jujube fruit development (Fig. 5b), and most of the genes were expressed at different levels. The genes of group IIe were mainly expressed at before white mature period (BWM) and white mature period (WM), and the expression of group IIb genes was lower at young fruit period (Y) except for ZjWRKY13. ZjWRKY8, 26, 47, and 48 were only involved in the development of young fruit, suggesting the role for these WRKY genes in jujube fruit development.
STRING analysis displayed that WRKY proteins could function by interacting with each other, as well as with MPK3 (Additional file 7A). Furtherly, yeast two-hybrid screening proved that ZjWRKY9 and ZjWRKY37 were interacting with ZjMAPKK6 (Fig. 6b), and ZjWRKY26 was interacting with ZjMAPKK2 (Fig. 6c). In previous study, it was found that ZjMAPKs and ZjMAPKKs were also involved in phytoplasma infection [28].
To confirm the identities of these ZjWRKY genes for phytoplasma tolerance, we analysed the transcript profiles of ZjWRKY genes in a JWB-resistant variety and a susceptible variety (Fig. 7). The detection of the phytoplasma in the two varieties was shown in Additional file 4. After phytoplasma infection, the expression of 9 ZjWRKYs (ZjWRKY2, 9, 22, 24, 29, 34, 36, 42, and 45) in the susceptible variety were higher than in the resistant variety, and they were also upregulated in above diseased tissues (Fig. 6). In contrast, the expression of ZjWRKY5 and ZjWRKY49 in the resistant variety was higher than in the susceptible one during the early stages of infection. These two genes were significantly downregulated in diseased tissues. ZjWRKY4 expression in the resistant variety was also higher than in the susceptible one. The above results indicated that some ZjWRKYs might play a role in phytoplasma tolerance.

Discussion
In this study, a total of 54 WRKY-encoding genes were identified in the jujube genome. These genes can be can be divided into three groups (Group I to III), but this number may increase in the future once problems with the assembly and annotation of the jujube genome are addressed. As in other plants, almost all of the ZjWRKY genes share the WRKYGQK signature motif. However, the WRKYGKK variant was found in two jujube genes ( Table 1, Fig. 2). Such slight variations in this region have also been reported in other plants such as Arabidopsis and apple [29].
Gene duplication events are the biggest contributors to the rapid expansion and evolution of gene families. Previous research has demonstrated that the Arabidopsis Group III WRKY gene family expanded rapidly as a result of recent segmental and tandem duplication events [30], and we found that this was also the case in the jujube genome. There are 6 tandemly duplicated ZjWRKY genes (ZjWRKY47, ZjWRKY49, ZjWRKY50, ZjWRKY52, ZjWRKY53, and ZjWRKY54) in Group III. The phylogenetic analysis (Additional file 5) indicated that 6 Group III ZjWRKYs were grouped and then clustered with 6 other genes from Arabidopsis; this also occurred in other subgroups from apple and pear. This finding suggests that the duplications in Group III WRKY genes occurred after the divergence of these plant species and tandem duplication events are the main contributors to the expansion of the Group III genes.
Previous research has demonstrated that Group I WRKY genes are the ancestors of the other WRKY genes in plants and are more likely to be constitutively expressed in different tissues [30]. In our study, the  Group I genes and many genes from the other two groups were expressed in various tissues (Fig. 5), indicative of their diverse functions. These results provide some useful clues for additional investigations into the biological functions of these WRKY genes in jujube growth and development. Transgenic apple lines overexpressing MdWRKY9 were significantly shorter and had significantly lower internode lengths than control plants [31], and its two orthologues in Arabidopsis (AtWRKY11 and AtWRKY17) are negative regulators of basal resistance to a bacterial pathogen [32][33][34]. The Group II phylogenetic tree (Fig. 8) indicates that AtWRKY11, 17, and ZjWRKY34 are closely related. In this study, we found that ZjWRKY34 was expressed at a noticeably higher level in infected jujube (Fig. 6) and in the JWB-resistant variety than in the susceptible variety at later stages of infection. STRING analysis showed that ZjWRKY34 (the orthologous of AtWRKY17) can interact with MSK1 and calmodulin (CAM, Fig. 9b). MKS1 is a regulator of plant defense response and it may contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors. It also indicated that ZjWRKY34 might interact with the calmodulin-Ca 2+ complex. Inferring the potential functions of ZjWRKY34 from the known AtWRKYs suggests that ZjWRKY34 might also act as a negative regulator in the defence process during jujube-phytoplasma interactions.
ZjWRKY9 was actively expressed in JWB-diseased tissues. The phylogenetic tree (Fig. 8) and sequence alignment showed that ZjWRKY9 was the orthologous gene of AtWRKY18. Transgenic AtWRKY18 plants had increased expression of pathogenesis-related genes and resistance to the bacterial pathogen Pseudomonas syringae, indicating that AtWRKY18 can positively modulate defence-related gene expression and disease resistance [12]. AtWRKY18/40 act in a feedback repression system controlling basal defences [10]. In the other side, high AtWRKY18 expression can cause severely abnormal plant growth [12]. These results suggest that proper expression of ZjWRKY9 is critical for enhancing jujube's defence response without negatively impacting plant growth. ZjWRKY9's higher expression might be related to the abnormal growth of diseased jujube trees, such as witches' broom and tiny leaves.
Overexpression of AtWRKY28 and AtWRKY75 induced an oxidative burst in host plants, which suppressed the hyphal growth of Sclerotinia sclerotiorum and consequently inhibited fungal infection [35]. STRING analysis predicted that AtWRKY75 could interact with GSTU10 (Fig. 9), which can eliminate the toxicity of oxygen bursts on plant cells and increase plant tolerance [36]. In this study, ZjWRKY22 and ZjWRKY24 are orthologous of AtWRKY75 (Fig. 8), which behaved higher expression in the susceptible variety than in the resistant one (Fig. 7). That means that the two genes might eliminate the toxicity of oxygen bursts caused by phytoplasma infection. Previous study also indicated that the expression of WRKY gene was responsive to phytoplasma infection [37]. A divergent behaviour was previously observed for OsWRKY28. In rice, overexpression of OsWRKY28 enhanced susceptibility to the rice blast fungus Magnaporthe oryzae and decreased accumulation of PR5 [38]. The knock-out of OsWRKY28 led to a two-fold increase in resistance to a compatible rice blast fungus and this phenotype is accompanied by the increased expression of several defence-related genes [39]. Hence, OsWRKY28 acts as a negative regulator of basal defence responses. Similarly, some WRKY genes might act as negative regulators of the basal resistance of jujube under phytoplasma stress, but further study is necessary to verify their specific functions.

Conclusions
This paper described the WRKY gene family of Chinese jujube at the genome level. Their gene structure, chromosomal distribution, phylogenetic relationship, and tissue-specific expression patterns were presented in this study. Most of the ZjWRKYs were positive responses to phytoplasma invasion, and that provided meaningful candidates for the future studies of ZjWRKYs involved in jujube-phytoplasma interaction.

Plant material
The seven tissues including roots, young branches, old branches, leaves, flower buds, flowers and young fruits were collected from three jujube trees and used for organ-specific expression analysis. Four kinds of tissues representing different degrees of JWB disease (apparently normal leaves (ANL), phyllody leaves (PL), and witches'-broom leaves (WBL)) from diseased trees, and healthy leaves (HL) from healthy trees were collected at four growth periods (June, July, August, and September). The All treatments were conducted with three biological replicates.
Phytoplasma cannot be cultured in vitro, and thus, JWB phytoplasma infection was transmitted by grafting. A JWB-resistant variety and a susceptible variety were used as scions for grafting onto JWB-diseased and healthy trees. All grafting treatments were conducted with three replicates. The samples were collected from sprouted scions at five growth periods (June, July, August, September and October). The samples were stored at − 80°C until RNA extraction and expression analysis.
The JWB phytoplasma presence of the samples was detected by quantitative real-time PCR (qRT-PCR) [40]. The expression of phytoplasma TMK gene in jujube samples was analysed and ZjACT was used as an internal control.

Identification and protein structure analysis of ZjWRKYs in Chinese jujube
First, WRKY genes from Arabidopsis were used as queries to search the jujube genome database. Next, the Pfam (http://pfam.xfam.org/) and SMART (http://smart.emblheidelberg.de/) databases were used to confirm the predicted jujube WRKY proteins. To further confirm that the amino acid sequences in our data set were WRKYs, we manually examined the conserved WRKYGQK amino acid motif at the N-terminus and the zinc-finger-like motif at the C-terminus of the predicted WRKY domain. Truncated and false genes were excluded from our analysis. The number of amino acids, molecular weight, and theoretical pI of ZjWRKY genes were predicted by Protparam (https://web.expasy.org/ compute_pi/). The conserved motifs of ZjWRKY proteins were detected by MEME (http://meme-suite.org/), using the following parameters: number of repetitions, any; maximum number of motifs, 20; and the optimum motif widths, 6-60 amino acid resides [41].

The chromosomal location and gene structure of ZjWRKYs
To determine the chromosomal location of the ZjWRKY genes, their gene sequences were used as query sequences in BLASTN searches against the jujube genome. Each ZjWRKY gene was mapped to the jujube genome according to their genome coordinates. Tandem duplications were identified as previously described [42]. The website GSDS (http://gsds.cbi.pku.edu.cn/) was used to predict the number of exons from the coding domain sequences (CDS) and DNA sequences of the WRKY genes [43].

Multiple sequence alignment and phylogenetic tree construction
The jujube WRKY proteins were classified into different groups based on their conserved domains. A phylogenetic tree was constructed from the amino acid sequences of WRKY conserved domains from jujube (54 sequences). The Arabidopsis thaliana WRKY proteins were retrieved from the TAIR database (http://www.arabidopsis.org/) as reported previously. Additionally, WRKY proteins of three other species (Persica prunus [22], Pyres bretschneideri [21], and Malus domestica [29]) were downloaded from NCBI. The classification of jujube Group II WRKY genes using the phylogenetic tree was dependent on the putative Arabidopsis thaliana orthologs. The MEGA 5.2 software and the neighbour-joining statistical method were used to construct a rooted phylogenetic tree [44][45][46]. The evolutionary distances were obtained using the p-distances method, and these distances were used to estimate the number of amino acid substitutions per site. The reliability of each phylogenetic tree was established by conducting 1000 bootstrap sampling iterations.

RNA isolation and expression and statistical analysis
Total RNA was extracted using an RNAprep Pure Plant Kit (TIANGEN) according to the manufacturer's protocol. After genomic DNA was removed by RNase-free DNase I (TIANGEN), RNA concentration and purity were checked on a NanoDrop2000 spectrophotometer. First-strand cDNA was synthesized by reverse transcribing 500 ng of total RNA with FastQuant RT Super Mix Kit (TIANGEN). The cDNA was used as the template for qRT-PCR. Gene expression was detected by qRT-PCR. The primers used in this study are listed in Additional file 6.
PCR products were amplified in triplicate using the Bio-Rad iQ™5 with TransStart Top Green qPCR Super-Mix AQ131 (TransGen Biotech, China) in 20 μL reactions. Each reaction contained 10 μL of 2 × TransStart® Top Green qPCR SuperMix, 0.4 μL each of 10 μM primers, 8.2 μL of ddH 2 O and 1 μL of cDNA. The thermal profile for RT-qPCR was as follows: preincubation for 30 s at 95°C, followed by 40 cycles of 5 s at 95°C,


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Germination of spores of Amazonian ferns Polypodium aureum in different culture media

The greatest diversity of ferns occurs in the tropics, where approximately three in four of the species are found. In the Brazilian flora, the ferns have megadiversity, being an important plant group. The objective was to evaluate the spores germination of ferns Polypodium aureum in different types of culture medium . The sporangia were removed from fertile fronds and sterilized with sodium hypochlorite 0.57% during 30 minutes. After sterilization, the sporangia were seeded in culture media agar, MS, SH, White and B5. The experimental design was completely randomized being composed of 5 treatments (agar media, B5, SH, White and MS) and 10 replications. The ratings occurred every 3 days over a period of 45 days, counting the number of spores germinated. After this period, the germination percentage, germination time on medium, germination speed index and timing of germination were calculated. The germination percentage and the index of synchronization have not changed in function of different culture media. The medium White presented the highest average for the germination speed index whereas the agar media, MS, SH and B5 had the same germination speeds. The percentage of mortality was higher in MS medium, the White medium presented the lowest percentage of mortality, and the media composed by agar, SH and B5 showed intermediate values. The lowest average germination time occurred at the media SH and B5, since the environment composed by agar showed higher average germination time. The media MS and White showed similar results to the agar, SH and B5. Based on the obtained results, it can be concluded that the White medium is the most effective on the germination of spores of Polypodium aureum . da germinação. A porcentagem de germinação e o índice de sincronização não alteraram em função dos diferentes meios de cultura. O meio White apresentou a maior média para o índice de velocidade de germinação já os meios ágar, MS, SH e B5 tiveram velocidades de germinação iguais. A mortalidade foi mais alta no meio MS, o meio White apresentou a menor mortalidade, e os meio compostos por ágar, SH e B5 apresentaram valores intermediários. O menor tempo médio de germinação ocorreu nos meios SH e B5, já o meio ágar apresentou maior tempo médio de germinação. Os meios MS e White apresentaram resultados semelhantes ao meio ágar, SH e B5. Com base nos resultados obtidos, conclui-se que o meio White é o mais efetivo na germinação dos esporos de P. aureum . Esporângios, sincronização, mortalidade, Pteridófitas.

Introduction
The Amazonian ferns, Polypodium aureum Lowe, are native from Brazil, from Polypodiaceae family, are perennial, vigorous herbaceous vegetations, and with rhizomes. It has large rhizomes and follicles, with large and crooked fronds (compound leaves). These species of ferns can be grown in pots containing good soil fertility and enriched with organic matter, are a little colder tolerant (Lorenzi et al., 2008). This ornamental species is mainly marketed and the information on its propagation is still very restricted.
The ferns of the species P. aureum can reproduce in two ways, the first way is by cuttings and the second way is by germination of spores. The cutting is a technique for vegetative propagation, which consists of stem and root segments planting of the plant matrix, giving origin to a new plant, from the rooting of the same. This method is widely used in the creations of seedlings of plants, especially trees and ornamental plants (Hartmann et al., 2002).
The in vitro cultivation of spores of P. aureum, is a very efficient method, because it allows the understanding of the life cycle of these plants and decreased the time of cultivation of the same, however, to obtain a culture without any kind of contamination, it is necessary that the spores be sterilized using sodium hypochlorite. (Pence, 2000).
In some countries, spores of P. aureum and other species of the same genus are being studied for medicinal purposes. (House et al, 1991;Das and Einstein, 2007). Hires (1940) made the first studies on in vitro culture with ferns, inoculating spores of P. aureum in solid nutrient medium; for the realization of the in vitro culture, different culture media can be used, but the P. aureum L., prefer culture media with low concentrations of nutrients.
It is only through the understanding of causal factors for genetic diversity that the speciation and evolution of pteridophytes can be understood and these factors are best observed through the sexual propagation obtained by spores (Lloyds, 1974).
The method presented here, is important because generates genetic variability, and by being an ornamental plant, individuals with characteristics of higher commercial value can be generated, adding value to the plant which may turn the activity more profitable. Despite the wide popular use, P. aureum presents few scientific studies. Therefore, this work had as objective to study the germination and survival of spores of P. aureum, in different culture media.

Material collection, general method of germination and sterilization
Fertile fronds of Polypodium aureum cultivated for commercialization were collected in a region that climate is AW according to the Koppen classification (22º07'04'' S; 51º22'04'' W and altitude of 430 m). The fronds were collected during the months of August and September 2016.
The Sporangia were removed from the fronds using a brush with stiff bristles, placed approximately 30 mg of Sporangia within a 3 mL syringe to facilitate the disinfection of the sporangia, then the sporangia were sterilized in a solution of sodium hypochlorite 0.57% during 30 min, which were obtained from a commercial solution diluted to 20%. After disinfection, the sporangia were washed three times in distilled water and autoclaved and quickly inserted into culture media, in sterile environment (Randi and Crozier, 1991).
The experimental design was completely randomized being composed of 5 treatments, being composed by media of agar, B5 (Gamborg et al., 1968), SH (Schenk and Hildebrant, 1972), White (White, 1951) and MS (Murashige and Skoog, 1962) and 10 replications for each culture medium. The nutrient solutions were previously sterilized in an autoclave, for 20 minutes at 120 °C and were deposited in the covered petri dish. Each 30 mg of spores were homogeneously dispersed in 5 Petri plates. After being seeded in culture media, the petri dishes were covered and sealed with a film of polypropylene.
The spores' germination occurred in a germination chamber, simulating local environmental conditions, with a photoperiod of 16 hours at a temperature of 25 ºC ± 2 °C and white light with photon flux density of approximately 30 µmol m -2 s -1 .
The ratings occurred every 3 days over a period of 45 days, counting the number of germinated spores. In order to count the number of germinated spores, it was necessary to make three circles, at the petri plates, where there were more spores, the circles were made in the magnification of 2x magnifying binocular stereoscopic microscope, using a marker for transparencies of permanent ink. The spores germinated within the circles were photographed in the increase of 2.5x in magnifying binocular stereomicroscope and counted with the help of Microsoft Paint.
Based on counts of germinated spores, the calculations of speed of germination index (Silva and Nakagawa, 1995), average time of germination (Laboriau, 1983) and index of synchronization (Laboriau and Agudo, 1987) were performed. At the end of 45 days, the counting of spores that germinated and that died was carried out to determine the percentage of dead spores.

Statistical Analysis
The data from experiments following completely randomized, were subjected to the Shapiro-Wilk test to verify the data normality and the Levine test to check the homogeneity of variances between culture medium values. Then, the data were subjected to analysis of variance and when significant the means were compared by the Tukey test at 5% probability.

Results and Discussion
The percentage of germination of spores of Polypodium aureum has not changed in function of the different culture media ( Figure 1A). All culture media provided 100% germination of spores.  Murashige and Skoog, 1962;Schenk and Hildebrant, 1972;Gamborg et al., 1968;White, 1951). The bars represent the mean values for each culture medium and the respective error bars indicate the standard deviation. Means followed by the same letters were not significantly different according Tukey test at 5% of confidence.
The viability of spores is directly related to the amount of reserve that the same show (Dyer, 1979) and these organic substances are essential for germination that come from the metabolism of the reserves of the same (Raghavan, 1980). The results of this study indicate that spores of P. aureum have some reserve, since they present good germination.
When they come in contact with water or even in a simple saline solution the spores of P. aureum are able to germinate, this occurs because the spores have in their interior reserve substances, this substance helps in the germination of spores (Raghavan, 1980 andMehltreter et al., 2010).
The time required for maximum germination of spores was different in function of the culture media ( Figure 1B). The medium composed by agar showed the greatest amount of days so that 100% of the spores germinated since that the SH medium was responsible for 100% germination of spores in only 35.5, and thus, a reduction of approximately 25% is observed. The media MS, B5 and White showed intermediate results with absolute values of 37.9, 38.4 and 42.3 days, respectively, so that 100% of the spores germinated, the latter, did not present statistical difference ( Figure 1B).
To control the growth of tissues and the development pattern of in vitro plants, it is necessary to use nutrient media that provide essential substances, such as macronutrients, micronutrients and vitamins (Pereira et al., 2003). However, these nutrients are not essential to the germination of spores (Soares et al., 2007).
The conditions used in this study favored the reproductive success of P. aureum spores. Such conditions influence the germination percentage and speed, influencing the rate of absorption of water and biochemical reactions that determine the whole process. Due to being exposed to these favorable conditions the process of germination of spores occurs with greater efficiency (Marcon et al., 2014).
The culture media influence the diffusion of water and nutrients for the plants, but if the environment is very consistent they may limit this function (Caldas et al., 1998). According to the same author, the solid media are more efficient, providing an optimal supply of salts for plants. Some culture media are formulated containing high concentrations of salts, however, culture media with high concentrations of salts hamper the in vitro development, causing stress, because the osmotic potential is reduced. Then the water is removed osmotically from the saline solution, increasing the concentrations of salts and consequently becoming less available to plants (Ribeiro, 2001). Excess nutrients cause damage by salinity, leading to an osmotic imbalance, negatively affecting the absorption of water by roots (Schossler et al., 2012). Probably for this reason the MS medium, although containing the highest amount of nutrients, was the one that presented higher spore mortality (Figure 2A).
Media with lower concentrations of nutrients provide a better absorption of water by the spores and consequently the spores can germinate, since water is a requirement to start the germination process (Whittier, 1970).
The SH culture medium had the lowest number of days for maximum spore germination ( Figure 1B). According to Spangenberg (2010) the SH medium is a basic culture medium formulated for the culture of tissues, organs, and cells. A basic culture medium is the one that provides essential substances for plants, such as, for example, water, macronutrients and micronutrients, vitamins, among others (Puignau, 1996). According to the same author, the pH of the culture media varies according to the species to be studied and with the culture medium, however all culture media must be corrected for pH= 5.7.
Due to being a basic medium this may have influenced the number of days for obtaining the maximum germination of spores. Before soaking, the spores present in their interior a very negative water potential, so when in contact with water, there is a rapid absorption and a rapid increase in the water potential of the embryo, due to the difference between potential existing between both, then there is a reduction in the water absorption, thus reducing the rate of hydration (Barrs and Weatherley, 1962). This entire process causes the expansion and cell division (Labouriau, 1987;Kermode, 1995).
The different culture media made the speed index of spores germination to be different ( Figure 1C). The mean value observed for GVI was 29.93, ranging from 29.05 to 42.72. The medium composed by White presented the highest average (42.72) for IVG, whereas the media Agar, MS, SH and B5 had equal speeds of germination (29.05), their germination occurred more slowly comparing to the White ( Figure 1C).
To calculate the germination rate index (IVG), one must calculate the data from the count of germinated plants; these calculations should establish the difference in speed of spores' germination (Biasi et al., 2009).
The culture medium White is composed of macronutrients and micronutrients in its composition. The macronutrients and micronutrients are required for the in vitro plants, because they have a heterotrophic metabolism (PIERIK, 1990). The amount of nutrients present in each culture medium influences the speed of spores' germination. Culture media that have smaller quantities of nutrients cause the spores to absorb more water, germinating faster.
A less concentrated culture medium is characterized by presenting a high water potential, this high water potential causes the spores to absorb more water from the environment through reverse osmosis and consequently they germinate more quickly. The water moves toward the regions of low water potential or low free energy, in order to equalize the concentrations. More concentrated culture media, i.e., with a high amount of nutrients, have a low water potential, causing the spores to take more time to moisturize and as a consequence the speed of germination delay (Whittier, 1975).
The amount of nutrients found in different culture media influence on mortality of gametophytes (Figure 2A). The percentage of mortality was higher in MS medium, approximately 37.28%. Whereas the White medium presented the lowest percentage of mortality, approximately 13.07%, in relation to other culture media. The medium composed by Hagar, SH and B5 showed intermediate values with absolute values of 19.2%, 27.99%, 24.07%, respectively.
The White' culture medium (1942) was one of the first basic medium to be formulated for the plants, showing low levels of nitrogen and potassium, even having in its formulation, low concentrations of salts, restricting its use, this medium is still widely used in various situations (Caldas et al., 1998). How it was to observed, the White' medium presented higher IVG ( Figure 1C) and lower mortality (Figure 2A).  Murashige and Skoog, 1962;Schenk and Hildebrant, 1972;Gamborg et al., 1968;White, 1951). The bars represent the mean values for each culture medium and the respective error bars indicate the standard deviation. Means followed by the same letters were not significantly different according Tukey test at 5% of confidence.
According to Caldas (1998) the White medium is a basic medium, i.e., provides essential substances for plants, which is characterized by having lower amounts of mineral salts, the cells of some plants prefer this culture medium, because they contain lower amounts of mineral salts, causing a lower percentage of mortality in the White medium.
Higher quantities of nutrients slow the germination process, causing necrosis and the growth of gametophytes is inhibited, this happens due to the high concentration of ammonia present in this environment (Fernández et al., 1997). The excess nutrients cause a nutritional disorder in the plants, these excess nutrients can cause deficiency of other essential elements for the plant, occurring a competition (Audebert and Fofana, 2009).
Due to the fact that the medium contains high levels of minerals, this may have influenced the increase of mortality of spores. Culture media containing large quantities of mineral salts interfere in the water potential of culture medium decreasing the water potential between the culture medium and the spores surface, this makes the spores not capture water, and increasing the mortality rate (Lopes et al., 2008).
The lowest average germination time occurred at 26 days, at the media SH and B5, since the environment composed by agar showed higher average time of eleven days longer than SH and B5.
The average time of germination is used to evaluate the speed at which a species occupies a given environment (Ferreira et al., 2001). According to Silveira et al. (2013), the average germination time was influenced by the nutrients found in each culture medium, when the spores were placed in the medium with large quantities of nutrients, there was a delay in the germination process, but when placed in media with lower concentrations of nutrients, the spores germinated more quickly, because, they managed to absorb more water and, consequently, initiating the germination process. Thus, this indicates that smaller quantities of nutrients promote the acceleration of germination of spores of P. aureum. Spores that have large quantities of water have a lower average germination time, when compared with spores with lower water contents (Dyer, 1979).
The media composed of agar, MS, SH, B5 and White provided the same timing of germination of spores ( Figure  2C). The synchronization indices are interpreted in a simple way, the lower the value the more synchronized the germination is in relation to time and the greater the value is the less synchronized the germination is (Duarte et al., 2015).
The synchronized germination of spores is beneficial, because the spores were able to reach the same stage of development in a short period of time.
When the index of this synchronization has low value means that the system is more orderly, with larger amounts of information, this causes increase of the values of percentage and speed of germination what occurs at the optimum temperature for germination (Labouriau and Osborn, 1987). In some culture media the germination of spores occurred quickly, whereas in other media the germination occurred slowly, but the germination of spores occurred at the same time.
The decrease of the average time of germination and the index of synchronization means that the different concentrations of nutrients influenced differently in water potential of culture media and consequently decreased the time of germination and the spores germinated in less time and in a more synchronized way.

Conclusion
The culture media agar, White (1951), MS (Murishige and Skoog, 1962), through B5 (Gamborg et al.,1968) and SH (Schenk and Hildebrant, 1972), were efficient, providing 100% of the germination of spores. However, the White medium is the most effective on the germination of spores of Polypodium aureum (Amazonian fern), providing a better germination speed and with low mortality rates.


extfieldsofstudy: Medicine Biology

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Identification of the Genes Involved in Anthocyanin Biosynthesis and Accumulation in Taxus chinensis

Taxus chinensis is a precious woody species with significant economic value. Anthocyanin as flavonoid derivatives plays a crucial role in plant biology and human health. However, the genes involved in anthocyanin biosynthesis have not been identified in T. chinensis. In this study, twenty-five genes involved in anthocyanin biosynthesis were identified, including chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, anthocyanidin synthase, flavonoid 3’-hydroxylase, flavonoid 3’,5’-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin reductase, and leucoanthocyanidin reductase. The conserved domains and phylogenetic relationships of these genes were characterized. The expression levels of these genes in different tissues and different ages of xylem were investigated. Additionally, the anthocyanin accumulation in xylem of different ages of T. chinensis was measured. The results showed the anthocyanin accumulation was correlated with the expression levels of dihydroflavonol 4-reductase, anthocyanidin synthase, flavonoid 3’-hydroxylase, and flavonoid 3’,5’-hydroxylase. Our results provide a basis for studying the regulation of the biosynthetic pathway for anthocyanins and wood color formation in T. chinensis.

Introduction
Secondary metabolites are the products of plant adaptation to the environment during their evolution. Anthocyanins as a kind of secondary metabolite play a vital role in various processes of plant development, such as providing color to organs to attract pollinators, and of antioxidant activity to protect plants from injury by biotic and abiotic stress [1][2][3][4]. In addition, anthocyanidins are widely used in food, health care products, and drugs for their excellent antioxidant activity [5,6]. Anthocyanins can be divided into different types according to the different chemical groups on the lateral chains of the anthocyanidin skeleton. Typical anthocyanins have the anthocyanidin skeleton called 2-phenylbenzo-pyran that contains an aromatic benzene ring-oxygenated heterocycle-aromatic benzene ring (C6-C3-C6) structure [7,8]. Cyanidin, pelargonidin, and delphinidin are the three most fundamental anthocyanidins in plants as all other anthocyanidins are directly or indirectly modified from these three types.
The structure and biosynthesis pathways of anthocyanins have been well studied. The genes related to anthocyanin biosynthesis have been reported in many plant species such as Arabidopsis thaliana, Medicago sativa, Petunia hybrida, and Salvia miltiorrhiza [9][10][11][12], which provide a good basis for understanding anthocyanin biosynthesis in these species. The biosynthesis of anthocyanin is complex. The 4-phosphoerythritose from the pentose phosphate pathway and the phosphoenolpyruvate from glycolysis are condensed into 7-phosphate heptanolose, which is modified into tyrosine and T. chinensis is a valuable woody species with important medicinal value. Paclitaxel, which is extracted from the bark of T. chinensis, is a natural antineoplastic drug that is widely used in the treatment of breast, ovarian, and lung cancers. In addition, the wood of Taxus species has many attributes, for example, good aesthetic appearance, purple red brown colored heartwood, straight texture, and rich elasticity [13,14]. The heartwood of T. chinensis contains high levels of anthocyanins and flavonoids. However, the genes involved in anthocyanins biosynthesis in T. chinensis have not yet been reported. To understand the correlation between gene expression and anthocyanin accumulation, we performed a systematic identification and analysis of genes related to anthocyanin biosynthesis in T. chinensis. In this study, 25 genes involved in anthocyanins biosynthesis in T. chinensis were identified through bioinformatics analysis and the conserved domains and phylogenetic relationships of these genes were analyzed. The expression profiles of these genes in different tissues and in different ages of T. chinensis xylem was also investigated. Moreover, the correlation between gene expression and anthocyanins accumulation was determined. Our results provide a basis for studying the regulation mechanism of biosynthetic pathway of anthocyanins in T. chinensis.

Plant Materials
T. chinensis was planted in the greenhouse located at Chinese Academy Forestry, Beijing, China. Leaf, xylem, phloem, root tissue, and one to four-year-old xylem were sampled from ten-year-old trees and stored in liquid nitrogen for RNA isolation. The bark was peeled from the developing stem, the phloem tissue was scraped from inside of the bark, and the xylem tissue was collected from the peeled log. Samples were taken from 3 plants and stored in ultra-cold storage freezer (−80°C) until use. One-to four-year-old xylem were weighed accurately for HPLC analysis of anthocyanin content.

Identification of Genes Related to Anthocyanin Biosynthesis
In order to predict the genes related to anthocyanin biosynthesis in T. chinensis, all the anthocyanin biosynthesis-related genes of Arabidopsis protein sequences were obtained from GenBank and were used as a query to search for homologous genes against the assembly of transcriptome (PRJNA580323) in T. chinensis using the tBLASTn algorithm with an e-value cut-off of 10 −10 . The predicted genes were inspected manually and corrected by sequence alignment with the genes involved in anthocyanin biosynthesis identified from other plant species by the BLASTx algorithm.

Gene Analysis in Bioinformatics and Phylogenetic Tree Construction
The molecular weight (Mw) and theoretical isoelectric point (pI) of deduced proteins involved in anthocyanin biosynthesis were analyzed by the Compute pI/MW tool on the ExPASy server (http://web.expasy.org/compute_pi/). Conserved domains were searched using CD-search tool on NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) against the Pfam v31.0-16709 PSSMs database. Multiple amino acid sequence alignments were performed by Clustal Omega tool on EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo). Phylogenetic trees were constructed by MEGA software (Philadelphia, CA, USA) with the neighbor joining (NJ) method (molecular evolutionary genetics analysis version 7.0) [15]. The protein sequences used for the sequence alignment and phylogenetic analysis were retrieved from NCBI. The accession numbers of protein sequences are used for the sequence alignment listed in Supplementary Tables S2-S6.

Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR)
Total RNA was isolated from the tissues of T. chinensis using the EASYspin plant total RNA isolation kit (Aidlab RN38, Beijing, China). RNA integrity and quantity were determined by 1.2% agarose gel electrophoresis and NanoDrop 1000C spectro-photometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription was performed using the FastKing RT Kit (with gDNase) (TIANGEN, Beijing, China). The qRT-PCR are performed with SYBR ® rapid quantitative PCR Kit (KAPA KK4601, Pleasanton, CA, USA). Primers used for qRT-PCR are listed in Table S1. Tcactin was used as a reference gene as described previously [16]. Gene expression levels were calculated according to the 2 -∆∆Ct method for the different tissues and aged xylem samples [17,18].

HPLC Analysis of Anthocyanin Contents
The conventional extraction method of anthocyanin is solvent extraction. Hydrochloric acid can prevent the degradation of non-acylation anthocyanin in the extraction process, and ultrasonic waves can improve the extraction efficiency [19]. Total anthocyanin was extracted by the ultrasonic assisted method [20]. Briefly, 200 mg of xylem was ground into powder in liquid nitrogen; the powder was then transferred into 2 mL methanol with 1% HCl at 4 • C protected from light for 12 h. After sufficient mixing, 2 mL of ddH 2 O and 4 mL chloroform were put in the solution and then centrifuged at 12,000 g for 20 min after ultrasonic treatment with an ultrasonic extractor for 1 h. The LC-20AD system (Shimadzu, Japan) and phenominex lunar C18 (4.6 µm × 250 mm) was used to analyze anthocyanin content according to previous studies [21,22]. The standard curve was established with cyanidin-3-glucoside chloride (Solarbio, Beijing, China) by the same method. Anthocyanin contents were calculated according to the standard curve.

Identification of Genes Involved in Anthocyanin Biosynthesis in T. chinensis
In order to identify genes in the pathway of anthocyanin biosynthesis in T. chinensis, BLAST analysis of protein sequences of these genes in Arabidopsis against the assembly transcriptome data of the T. chinensis was performed using the tBLASTn algorithm [23]. A total of 25 genes were identified with 22 of these genes not previously identified. They belonged to nine families including CHS, CHI, F3'H, F3'5'H, F3H, LAR, DFR, ANR, and ANS. The identified genes are named TcCHS, TcCHI1-TcCHI2, TcF3'H1-TcF3'H4, TcF3'5'H, TcF3H1-TcF3H5, TcLAR1-TcLAR2, TcDFR1-TcDFR8, TcANR, and TcANS (Table 1). Sequence feature analysis of these genes included the length of ORFs (open reading frames), the molecular weight (Mw), the size of deduced proteins, and the theoretical pI which are listed in Table 1. All the deduced protein sequences contained the conserved domains ( Figures S1-S3), suggesting that they are the likely proteins involved in anthocyanin biosynthesis pathway in T. chinensis. Analysis of the deduced protein sequences against the Nr/nt database using BLAST algorithm showed that TcCHI2 has extremely high identities (98.58%) with TcCHI (AIQ85030.1, unpublished). TcF3'H4 and TcF3'5'H have 99.61% and 100% identity with CYP75B115 (ATG29929.1) and CYP75A77 (ATG29931.1) that were previously predicted from transcriptome data [24]. The other 22 genes were identified in this study.

Phylogenetic Analysis and Expression Pattern of TcCHS
CHS plays a crucial role in anthocyanin biosynthesis pathway, and it is also one of the decisive factors affecting anthocyanin content [25]. CHS is one member of the type III polyketide synthase (PKS) family with two conserved domains (Chal_sti_synt_N and Chal_sti_synt_C). The Chal_sti synt_C domain is similar to domains of thiolase and beta-ketoacyl synthase. Only one CHS was identified in T. chinensis. The TcCHS contained two conserved motifs ( Figure S1) and shared high sequence similarities with other plant species. Multiple protein sequence alignment of TcCHS and CHSs from other plant species indicated that all of them had the catalytic triad (Cys164-His303-Asn336) ( Figure S4), which is the core active site of the type III PKS. In addition, the "gatekeepers" Phe-215 and Phe-265 and some inert active site residues such as Thr-132, Ser-133, Thr-194, Thr-197, Gly-256, and Ser-338 are highly conserved in the plants used in this study, except for Thr-197 of PcCHS which is replaced by Cys [26,27]. Amino acid sequences of CHS from 15 plant species including dicots, monocots, and gymnosperms were used to conduct the phylogenetic tree. The phylogenetic tree indicated that the CHSs were clustered into three clades (Figure 2a). TcCHS was included in clade1 with GbCHS, GmCHS, MdCHS, AtCHS, CsCHS, MsCHS, PtCHS, VvCHS, TuCHS, OsCHS, and GaCHS. Clade1 included both angiosperms and gymnosperms CHSs, indicating the function of these CHSs is highly conserved and consistent with a previous study [12]. The expression levels of TcCHS in roots, leaves, phloem, and xylem were analyzed using qPCR (Figure 2b). TcCHS exhibited the highest expression level in xylem, followed by phloem and leaves, with the lowest in roots. This indicated that TcCHS may play an important role in the development of xylem. The expression level in roots was arbitrarily set to 1. Error bars represent the standard deviation of three technical PCR replicates. One-way ANOVA was calculated using IBM SPSS 19 software. p < 0.01 was considered statistically significant and was represented by asterisks.

Phylogenetic Analysis and Expression Pattern of TcCHIs
CHI generally catalyzes naringenin chalcone into the flavanone (2S)-naringenin. It can be divided into four types including type I, type II, type III, and type IV. We identified two putative CHI genes from T. chinensis. The TcCHIs contained the conserved domain Chalcone_3 (pfam02431) ( Figure S1). Multiple amino acid sequence alignments of TcCHIs and type I (AtCHI), type II (MsCHI), type III (AtFAP1), and type IV (AtCHIL) were performed [12,28,29] ( Figure S5). Due to TcCHI1 possessing two Chalcone_3 domains, we separated it into two fragments for the alignment. The result showed that the two fragments of TcCHI1 share high sequence identity with AtCHI, indicating that TcCHI1 belongs to a type I CHI. Doubling of the functional domain might be caused by gene duplication. TcCHI2 has high sequence identity with AtCHIL, indicating it belongs to a type IV CHI.
Amino acid sequences of CHI from plant species as previously used in CHS were used to conduct the phylogenetic tree except for Ginkgo biloba as no identified CHI from gymnosperms has been reported. The phylogenetic tree showed that the CHIs were divided into four clades ( Figure 3a) and that most of the CHIs belong to type I, which is consistent with two previous studies [29,30]. TcCHI1 and TcCHI2 were clustered with AtCHI (type I CHI) and AtCHIL (type IV CHI), respectively, which is consistent with the multiple sequence alignment. There are some differences in the four types of CHI with regards to their biological functions [28]. Analysis of the qRT-PCR data showed that the two TcCHIs were expressed in all tissues of T. chinensis but exhibited different expression patterns. TcCHI1 was highly expressed in leaves, followed by roots and xylem, and less in phloem, whereas TcCHI2 showed the highest expression in xylem, followed by leaves and roots, and lower in phloem ( Figure 3b).

Phylogenetic Analysis and Expression Pattern of TcF3Hs and TcANS
F3H and ANS contain two conserved domains, including DIOX_N (pfam03171) and 2OG-FeII_Oxy (pfam14226) ( Figure S1), and they belong to the 2-oxoglutarate dependent dioxygenase (2-ODD) superfamily. The DIOX_N domain is highly conserved in the N-terminal region with 2-oxoglutarate/Fe (II)-dependent dioxygenase activity [12,[31][32][33]. F3H catalyzes naringenin, eriodictyol, and dihydrotricetin into dihydrokaempferol, dihydroquercetin, and dihydromyricetin. ANS catalyzes leucopelargonidin, leucocyanidin, and leucodelphinidin into pelargonidin, cyanidin, and delphinidin. We identified five putative F3H genes and one putative ANS gene from T. chinensis. The deduced amino acid sequences were aligned with F3Hs and ANSs of other plant species; the result showed that all of TcF3Hs and TcANS have the conserved H-x-D-xn-H motif ( Figures S6 and S7) [34,35]. TcF3H1 and TcF3H2 have higher identity with AtF3H, whereas TcF3H3, TcF3H4, and TcF3H5 have higher identity with GbF3H. TcANS contains enzyme-specific active sites of ANS including Arg-298, which form the electrostatic interaction, and Phe-304, which binds the A-ring of the substrate and the "lip"structure (Val-235, Phe-334, IIe-338, and Leu-342) except for Val-235, which was replaced by Ile in T. chinensis. The expression level in roots was arbitrarily set to 1. Error bars represent the standard deviation of three technical PCR replicates. One-way ANOVA was calculated using IBM SPSS 19 software. p < 0.01 was considered statistically significant, and there was no significant difference between different tissues.
The phylogenetic tree was constructed by using amino acid sequences of F3Hs and ANSs from plant species that are partially different from CHS, as there was no identified F3H and ANS from the same plant species. The phylogenetic trees showed that all the TcF3Hs and TcANS are clustered with gymnosperm F3Hs and ANSs (Figure 4a). TcF3H1 clustered with F3Hs from Pinus radiata, Picea sitchiensis, and Picea glauca, whereas TcF3H3, TcF3H4, and TcF3H5 were clustered with F3H from Ginkgo biloba (Figure 4a), implying possible different roles. The analysis of expression revealed that TcF3Hs had different expression patterns in different tissues (Figure 4b). TcF3H1 showed similar expression levels in each tissue, whereas TcF3H3 and TcF3H4 showed prominently high expression levels in roots and phloem, respectively. In addition, TcF3H2 showed prominently low expression levels in xylem and roots. The expression level of TcF3H5 increased slightly in leaves, phloem, xylem, and roots. These results indicate that different TcF3Hs may have different functions in T. chinensis. TcANS exhibited high expression levels in phloem and xylem, implying that TcANS may be a candidate gene that participates in the color formation of red brown bark (Figure 4b).
3.5. Phylogenetic Analysis and Expression Pattern of TcF3'Hs and TcF3' 5'H F3'H and F3'5'H belong to the cytochrome P450-dependent monooxygenase superfamily and are haem-thiolate proteins involved in the oxidative degradation of various compounds [36,37]. They catalyze 3' or 3',5' sites of the benzene ring in dihydrokaempferol oxygenated into taxifolin or dihydromyricetin. Four possible TcF3'Hs and one TcF3'5'H gene were identified in T. chinensis. All the deduced proteins contained p450 domain (pfam00067) ( Figure S2), which belonged to the cytochrome P450 superfamily. The p450 domain usually contained conserved motifs including the proline-rich "hinge" region, oxygen binding pocket motif, E-R-R motif, and heme-binding domain [38,39]. Alignment of TcF3'Hs, TcF3'5'H and F3'Hs, F3'5'Hs from other plant species showed that the TcF3'Hs and TcF3'5'H contained the conserved motifs mentioned above ( Figures S8 and  S9). The proline-rich "hinge" region (PPGXXXP) is conserved at both ends, which is consistent with previous work [37]. The oxygen binding pocket motif (AGTDTSS) is almost completely conserved for F3'5'Hs but not for F3'Hs. It is replaced by "GGTESSA" for TcF3'H1, DcF3'H, and AcF3'H, while it is replaced by "AGTDTAS" and "GSTDTTS" for TcF3'H2 and TcF3'H3, respectively. Similar to the E-R-R motif and heme-binding domain, it is completely conserved for F3'5'Hs, whereas it has a one amino acid difference for F3'Hs. In addition, all of F3'5'Hs have the two conserved sequences "GHML" and "GLALQK" and F3'Hs do not. The differences in all these sequences may be responsible for the substrate specificity of the two enzymes [40].
Phylogenetic trees were constructed with F3'Hs and F3'5'Hs from plant species, of which F3'H and F3'5'H genes have been identified, including four TcF3'Hs and one TcF3'5'H ( Figure 5a). TcF3'H1-TcF3'H2 clustered together with F3'H of Pohlia nutans (lower plant), whereas TcF3'H3-TcF3'H4 clustered together with F3'Hs of Plectranthus scutellarioides (dicot). TcF3'5'H clustered together with F3'5'Hs of Glycine max and Salvia miltiorrhiza. TcF3'H1 showed higher expression level in roots than other tissues, while TcF3'H2 showed the lowest expression level in roots (Figure 5b). TcF3'H3 exhibited higher expression level in leaves and phloem than that in roots and xylem; TcF3'H4 exhibited lower expression level in xylem than that in leaves, phloem, and roots. This possibly reflects the functional diversity of TcF3'Hs in T. chinensis. TcF3'5'H exhibited lower expression level in leaves than that in phloem, xylem, and roots ( Figure 5b). The different spatial and temporal expression patterns of TcF3'Hs and TcF3' 5'H indicate possibly different roles in the growth process of T. chinensis. The expression level in roots was arbitrarily set to 1. Error bars represent the standard deviation of three technical PCR replicates. One-way ANOVA was calculated using IBM SPSS 19 software. p < 0.01 was considered statistically significant and was represented by asterisks.

Phylogenetic Analysis and Expression Pattern of TcDFRs and TcANR
DFR catalyze dihydroflavonols into leucoanthocyanidins, which are the substrates of ANS. The products of ANS have two possible outcomes: one is conversion into anthocyanins by chemical modification, and the other is the production of flavane-3-alcohol catalyzed by ANR. DFR and ANR belong to the NAD/NADH-dependent epimerase family, which use nucleotide sugar as substrate for a variety of chemical reactions [41][42][43]. All of TcDFRs and TcANR contained the conserved domain epimerase (pfam01370) ( Figure S3). DFR has three kinds of substrates including dihydrokaempferol, dihydroquerceti, and dihydromyricetin, which make contributions to different colors, in most plants, whereas DFR reduces the combining capability of substrates that cannot produce orange such as Petunia [44,45]. Multiple amino acid sequence alignments of GaDFR, MnDFR, GbDFR, VvDFR, AtDFR, and TcDFRs indicated that all TcDFRs have the enzyme specific active sites including Ser-128, Tyr-163, and Lys-167 (numbers refer to VvDFR) (Figures S10 and S11) [42]. There is a substrate specific recognition region between Ser-128 and Tyr-163 of which was reported that the conversion of Asn-133 to Asp-133 made the enzyme lose the combining capability of dihydrokaempferol as a substrate. TcDFR3 has Asn-133 in the substrate-specific recognition region alike that of GbDFR, VvDFR, and AtDFR, indicating that they can combine all the three substrates. Morus notabilis may lack the ability since MnDFR has Asp-133 in the recognition region. In addition, the other TcDFRs and GaDFR (Gossypium arboreum) have neither Asn nor Asp in the specific site, indicating that there are different substrate specific recognition mechanisms of DFR. Multiple amino acid sequence alignment of TcANR and ANRs from other species showed they were highly conserved. TcANR had active sites (Ser-130, Tyr-167, and Lys-171) and the NAD/NADH combination domain (G-G-X-G-X-X-A).
Amino acid sequences of DFR and ANR from plant species as previously used in CHS were used to conduct the phylogenetic tree, including eight TcDFRs and one TcANR, respectively (Figure 6a). It showed that TcDFR3 is closely related to GbDFR (Ginkgo biloba), which is consistent with the results of a previous study [45]. The other seven TcDFRs were closely related to angiosperm plants such as GaDFR (Gossypium arboreum), GmDFR (Glycine max), and NtDFR (Nicotiana tabacum) especially for TcDFR1, TcDFR2, and TcDFR5. TcANR clustered with PsANR (Picea sitchensis) and GbANR (Ginkgo biloba), which are gymnosperms. The analysis of expression profiles revealed that TcDFR5 exhibited similar expression in the tissues analyzed; TcDFR2 and TcDFR6 exhibited prominently high expression level in leaves, TcDFR3 and TcDFR7 exhibited mainly high expression level in roots and TcDFR1, and TcDFR4 exhibited high expression levels in phloem and xylem (Figure 6b). In addition, TcDFR8 had almost no expression in leaves, phloem, and xylem, except for roots. The expression level of TcANR exhibited no significant difference among the different tissues (Figure 6b). Together with the expression data, the conservation and diversity of TcDFRs indicate their possible important role in anthocyanin biosynthesis in T. chinensis. The expression level in roots was arbitrarily set to 1. Error bars represent the standard deviation of three technical PCR replicates. One-way ANOVA was calculated using IBM SPSS 19 software. p < 0.01 was considered statistically significant and was represented by asterisks. The expression level in roots was arbitrarily set to 1. Error bars represent the standard deviation of three technical PCR replicates. One-way ANOVA was calculated using IBM SPSS 19 software. p < 0.01 was considered statistically significant and was represented by asterisks.

Phylogenetic Analysis and Expression Pattern of TcLARs
LAR is a NADPH-dependent enzyme that catalyzes leucoanthocyanidin into (+)-flavane-3-alcohol, which is the precursor of procyanidin [46]. LAR has been identified and characterized by the conserved domain NmrA (pfam05368) in a number of plant species such as Vitis vinifera, Malus domestica, and Medicago truncatula, and the crystal structure from Vitis vinifera has been analyzed [47][48][49][50]. Ser-118, Lys-140, Ile-162, and Asp-98 are residues associated with NADPH binding. In this study, we identified two TcLARs. Sequence feature of TcLARs is shown in Table 1. The deduced TcLAR proteins contain the NmrA domain ( Figure S2) and Ser-118, Lys-140, and Ile-162 residues. It has been shown that Asp-98 is conserved in VvLAR, FtLAR, CsLAR, MrLAR, PaLAR, and PtLAR but is replaced by Asn, Glu, and Ser in CasLAR, TcLAR1, and TcLAR2 ( Figure S12). In addition, Tyr-137 and His-122 are associated with OH-7 binding site; Met-136, Val-269, and Phe-272 are associated with A-C ring binding site; and Gly-93 and Met-136 are associated with the B-ring binding site. TcLARs all have the enzyme active residues except Met-136, which is replaced by Phe, similar to PaLAR and PtLAR.
Phylogenetic trees were constructed with LARs from plant species of which LAR genes have been identified. The phylogenetic tree revealed that the LARs are divided into three clades: clade 1 (monocots), clade 2 (gymnosperms), and clade 3 (dicots) (Figure 7a). TcLARs and other gymnosperms LARs clustered together, which is consistent with the genetic classification of plants. The expression patterns of TcLAR1 and TcLAR2 are different in different tissues (Figure 7b). The expression levels of TcLAR1 in leaves, xylem, and phloem were significantly higher than that in root, while the expression level of TcLAR2 in roots was higher than that in other tissues, suggesting TcLAR1 and TcLAR2 may have different gene functions.

Correlation Analysis of the Expression of Genes Involved in Anthocyanin Biosynthesis and Anthocyanin Accumulation
In order to investigate the relationship between the expression levels of genes involved in anthocyanin biosynthesis and anthocyanin accumulation, the content of anthocyanin and the expression level of genes involved in anthocyanin biosynthesis of xylem ranging in age between one to four years in T. chinensis was measured by HPLC; 1% methanol hydrochloric acid was used as solvent in combination with ultrasonic extractor-assisted extraction at low temperature and dark conditions to obtain the best extraction. The results showed that one-year-old xylem had relatively higher anthocyanin content, which increased gradually from two-year-old to four-year-old xylem (Figure 8a). The chromatographic data is shown in Supplementary Table S8. The one-year-old xylem may contain more chlorophyll, covering up some red pigments that make the one-year-old branches appear green.
Expression levels of genes involved in anthocyanin biosynthesis in T. chinensis were analyzed using qRT-PCR (Figure 8b,c). The results showed that TcCHS exhibited higher expression levels in one-year-old xylem than any other sample. TcCHI1 and TcCHI2 both exhibit higher expression levels in one-year-old xylem than others, especially for TcCHI2. This may suggest that the enhanced effect on anthocyanin biosynthesis in one-year-old xylem is also higher than that in older xylem. TcF3H1, TcF3H2, and TcF3H3 exhibited higher expression levels in one-year-old xylem than older xylem; TcF3H4 and TcF3H5 exhibited similar expression level in the different ages of xylem, except for relatively high level of TcF3H4 in four-year-old xylem. TcF3'H2 and TcF3'H4 exhibited higher expression levels in one-year-old xylem and three-year-old xylem, respectively, while TcF3'H1 and TcF3'H3 exhibited higher expression levels in four-year-old xylem. TcF3'5'H exhibited higher expression level in one-year-old xylem than that in older xylem. TcDFRs exhibited similar expression patterns except for relatively high levels for TcDFR1, TcDFR4, and TcDFR8 in one-year-old xylem. TcANR exhibited similar expression level in each tissue and higher levels in one-year-old xylem than other ages. TcLAR1 showed similar expression, while TcLAR2 showed the highest expression in one-year-old xylem. Taken together, the anthocyanin accumulation was correlated with the expression levels of TcDFR1, TcANS, TcF3'H1, and TcF3'5'H, suggesting these genes may play a vital role in the anthocyanin biosynthesis of xylem. The expression level in roots was arbitrarily set to 1. Error bars represent the standard deviation of three technical PCR replicates. One-way ANOVA was calculated using IBM SPSS 19 software. p < 0.01 was considered statistically significant and was represented by asterisks.

Discussion
Anthocyanins are an important type of secondary metabolites which play significant roles in colored plant tissues, such as flowers, fruits, and wood. Although the genes related to biosynthesis of anthocyanin have been identified in many plant species, they have not been systematically identified in T. chinensis. In this study, up to 25 genes putatively related to anthocyanin biosynthesis were identified in T. chinensis, including TcCHS, TcCHI1-TcCHI2, TcF3H1-TcF3H5, TcF3'H1-TcF3'H4, TcF3'5'H, TcDFR1-TcDFR8, TcANS, TcANR, and TcLAR1-TcLAR2. Only three of these have been previously reported. The sequence features and tissue-specific expression patterns of these genes in T. chinensis were analyzed. The conserved domains of deduced amino acid sequences of these genes and their phylogenetic relationships were performed and found to have high identity and close phylogenetic relationships with the corresponding genes from gymnosperms.
T. chinensis is thought to have evolved in the Paleozoic era approximately 300 million years ago, surviving the harsh environment of the Quaternary glacier period. It belongs to relict plants of the Tertiary period and has a longer evolutionary period than angiosperms. Analysis of phylogenetic trees conducted in this study showed that most of the identified T. chinensis genes are located at the root of the phylogenetic tree, which is consistent with the evolutionary relationship between T. chinensis and other angiosperms [51,52]. However, different members in different gene families have different evolutionary relationships due to genetic differentiation [53]. Genes such as TcF3'H1 and TcLAR2 of which deduced proteins were closely related to angiosperms in phylogenetic trees may have more variation in their selection process. The gene family originated by gene duplication and divergence from a common ancestor. Interestingly, TcCHI1 possesses two Chalcone_3 domains in a tandem array in T. chinensis, whereas one CHI gene contains only one conserved domain similar to that found in other species. The sequence analysis of Lotus japonicus showed that there were four CHI genes in a tandem array in its genome [29]. Since T. chinensis precedes Lotus japonicus in the evolutionary relationship, we speculate that the ancestor gene of CHI family existed with several tandemly conserved domains and became a cluster of tandem genes through the long period of evolution. The number of genes identified in anthocyanin biosynthesis-related gene families in T. chinensis were different from other plant species such as Arabidopsis thaliana, salvia miltiorrhiza, and Oryza sativa, indicating that genes encoding the same enzyme may have different evolution patterns in different species (Table S7) [12,28,54], for example, the number of T. chinensis DFR genes is significantly more than the number in Arabidopsis thaliana, Salvia miltiorrhiza, and Oryza sativa, suggesting that more gene duplication events occurred for DFR genes in T. chinensis (Table S7).
The anthocyanin biosynthesis pathway has been extensively studied in plants. A number of studies about transcriptomics and metabonomics in anthocyanin biosynthesis revealed that the expression levels of structural genes play an important role in anthocyanin accumulation, especially DFR, ANS, and F3H [55][56][57][58]. DFR and ANS are considered as very important enzymes in anthocyanin biosynthesis, as they could direct the flavonoid flux into the anthocyanin branch [55][56][57][58]. Previous studies showed that the expression levels of DFR, ANS, and F3H and some transcription factors were positively correlated with anthocyanin accumulation. The expression levels of TcDFR1, TcDFR4, and TcDFR5 showed higher expression levels in phloem than in other tissues. Similarly, the expression levels of TcDFR3and TcDFR8 showed high expression in roots, possibility resulting in high anthocyanins accumulation in phloem and root. This is consistent with the red bark and red root of T. chinensis.
The wood of T. chinensis has high commercial value because of its aesthetic appearance, straight texture, high density, mechanical strength, rich elasticity, corrosion resistance, and purple red brown colored wood. The color of the wood is an important trait reflecting its quality and economic value. It has been shown that the formation of wood color is due to the existence of secondary metabolites in heartwood, including different kinds of phenolic compounds [59]. In order to study the effect of anthocyanin biosynthesis in wood formation, analysis of expression patterns of genes related to anthocyanin biosynthesis and the accumulation of anthocyanins in differently aged xylem of T. chinensis was conducted. Analysis of the expression levels of these genes in different tissues showed different expression patterns, suggesting potential different functions. The expression levels of TcCHS, TcANS, and TcDFR1 showed relatively higher in xylem than in other tissues. The expression levels of TcDFR1, TcANS, TcF3'H1, and TcF3'5'H were observed to increase gradually from two-year-old to four-year-old xylem, especially for TcDFR1 and TcF3'5'H that showed the highest expression level in one-year-old xylem, which is consistent with the trend of anthocyanin content accumulation (Figure 8a,b). In the flavonoid biosynthetic pathway, CHS, CHI, F3H, F3'H, and F3'5'H catalyze the flavonol biosynthesis, whereas DFR, ANS, and ANR lead to anthocyanin and procyanidins biosynthesis. F3'H and F3'5'H play primary roles in the diversification of anthocyanins through determining their B-ring hydroxylation pattern. DFR is specific for substrates which affect the anthocyanin composition and pigmentation [60]. ANS is a key enzyme for anthocyanin biosynthesis, which catalyzes the conversion of leucoanthocyanidin into anthocyanidin [61]. Our results indicate that the accumulation of xylem anthocyanins in T. chinensis has a close association with TcDFR1, TcANS, TcF3'H1, and TcF3'5'H their expression levels not only are consistent with the trend of anthocyanin content but also showed relatively high expression levels in xylem (Figure 8b,c), implying their potential important roles in anthocyanin accumulation in xylem, which provides a framework for future research focused on improved wood color. Another noteworthy result was that 14 of all the 25 genes exhibited the highest expression level in one-year-old xylem, which had the highest content of anthocyanins. This may be because one-year-old xylem as a young tissue has very high level of primary metabolism, which can provide the raw material for secondary metabolism. These results provide insights into the genes associated with wood color formation. Our results provide a basis for further research and possibly the manipulation of the regulation and accumulation of anthocyanins in T. chinensis.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/2073-4425/10/12/982/ s1. Table S1. Primers used for qRT-PCR; Table S2. The proteins used in the alignment and of CHS and CHI; Table S3. The proteins used in the alignment of F3H and ANS; Table S4. The proteins used in the alignment of F3'H and F3'5'H; Table S5. The proteins used in the alignment of DFR and ANR; Table S6. The proteins used in the alignment of LAR; Table S7. Anthocyanin biosynthesis genes identified in different plant species; Table S8. Chromatographic data of anthocyanin content determination; Table S9. CDS and AA sequences of Anthocyanin Biosynthetic Enzyme Genes in Taxus chinensis; Figure S1. Conserved domains of TcCHS, TcCHI1-TcCHI2, TcF3H1-TcF3H5, and TcANS; Figure S2. Conserved domains of TcF3'H1-TcF3'H4, TcF3'5'H, and TcLAR1-TcLAR2; Figure S3. Conserved domains of TcDFR1-TcDFR8 and TcANR, Figure S4. Multiple sequence alignment of TcCHS against CHSs from other plant spices; Figure S5. Multiple sequence alignment of TcCHI1-TcCHI2 against CHIs from other plant spices; Figure S6. Multiple sequence alignment of TcF3H1-TcF3H5 against F3Hs from other plant spices; Figure S7. Multiple sequence alignment of TcANS against ANSs from other plant spices; Figure S8. Multiple sequence alignment of TcF3'H1-TcF3'H4 against F3'Hs from other plant spices; Figure S9. Multiple sequence alignment of TcF3'5'H against F3'5'Hs from other plant spices; Figure S10. Multiple sequence alignment of TcDFR1-TcDFR8 against DFRs from other plant spices; Figure S11. Multiple sequence alignment of TcANR against ANRs from other plant spices; Figure S12. Multiple sequence alignment of TcLAR against LARs from other plant spices.

Conflicts of Interest:
The authors declare that they have no competing interests.