Background

Lily is one of the world-famous fresh cut flowers, among which Oriental lily is the most common variety and widely favored due to its advantages of large flowers, graceful shape and rich flavor. However, single color is the main defect of Oriental lily in the application of flower cutting. Studies on the function of key enzyme genes in lily carotenoid metabolism pathway can not only further explore the carotenoid metabolism pathway of lily, but also research the coloring mechanism of lily and apply it to molecular breeding, which has important practical significance for achieving the cultivation of new varieties of lily (Huang et al., 2012).

There are three main types of pigments in higher plant leaves: chlorophyll, carotenoid and flavonoid pigments (mainly anthocyanins). Carotenoid can not only play an important role in the color of plants, but also absorb and transfer light energy, protect chlorophyll and delay the rapid decomposition of chlorophyll in leaf photosynthesis (Xia et al., 2013; Guo et al., 2015; Kong et al., 2015; Lu and Liu, 2016), which is an indispensable structural component of photosynthetic antenna and reaction center complex. In addition, under the regulation of three mechanisms (synthesis, degradation and storage), the accumulation of total carotenoid content is closely related to it. If gene mutation occurs in a certain mechanism, the coloring of plants will mutate (Lu and Liu, 2016).

Carotenoids are a kind of important fat-soluble pigment population widely distributed in nature, including C40 or C30 terpenoids with isoprene skeleton (Johnson, 2009). Since the separation of carotenoids in the early 19th century, nearly 800 kinds of natural carotenoids have been found (Guo et al., 2015). The biosynthesis of carotenoids includes series of condensation, dehydrogenation, cyclization, hydroxylation, epoxidation, etc. (Isaacson et al., 2002; Park et al., 2002). The cyclization of lycopene is catalyzed by LCYB and LCYE, the key branch point of carotenoid metabolism pathway. LCYB and LCYE are important cyclases to form β-carotenoid and ε-carotenoid, the amino acid sequence homology of which reaches 35% (Cunningham and Gantt, 2001).

There are no related reports about key enzyme gene LCYB of carotenoid metabolism pathway in lily. In this study, the key enzyme gene LCYB in cloning metabolism pathway and related bioinformatics analysis were carried out in order to provide theoretical basis for further exploring the metabolism pathway and the role of carotenoid in chromaticity.

1 Results and Analysis

1.1 Determination of carotenoid and chlorophyll content

It was found that the contents of carotenoid and chlorophyll were significantly different in seven parts (stigma, anther, petal, leaf, stem, bulb and root) of Oriental lily ‘Justina’ (Figure 1). The variance analysis was carried out for the content of carotenoid and chlorophyll in different parts by Spass 17.0. The results showed that the total content of carotenoid and lutein in anthers and leaves were significantly higher than those in other parts. The total content of carotenoid and lutein in anthers were the highest. The content of chlorophyll a in leaves, stigmas and stems were higher, but the content of chlorophyll a in leaves was significantly higher than those in stigmas and stems. The content of chlorophyll b in leaves and stems were higher, and the content of chlorophyll b in leaves was significantly higher than that in stems. The content of carotene in anthers and leaves were higher, which was significantly higher than those in other parts.

Oriental lily ‘Justina’ with about 10 cm stem growing from bulb was used as experimental material and the leaves were collected at 10 d, 25 d, 50 d, 75 d and 100 d, respectively. The trends of the relative content of chlorophyll and carotenoid in leaves were measured (Figure 2). The results showed that the relative content of chlorophyll a, chlorophyll b, total carotenoid, carotene and lutein in lily leaves increased gradually along with time from day 10 to day 100. Among them, the relative content of chlorophyll an increased most remarkably over time, the rising trend of chlorophyll b, carotene and lutein was relatively gentle, and the relative content of lutein remained about the same during 50-100 d. The content of chlorophyll a, chlorophyll b, total carotene and carotenoid increased significantly within 75-100 d, and the growth trend of chlorophyll a was the most obvious.

1.2 Semi-quantitative expression analysis of key enzyme genes in carotenoid metabolism pathway of lily

In 5 stages and 7 different parts of lily leaf growth, the semi-quantitative expression analysis was carried out on 12 key enzyme genes of PSY, PDS, Z-ISO, ZDS, CRTISO, LCYB, LCYE, HYB, VDE, ZEP, CCS and NCED in carotenoid metabolism pathway (Figure 3). According to the results, compared with the expression level of 18S rRNA, the expression level of PSY, Z-ISO, CRTISO, HYB and CCS genes in the leaves of Lily remained at a lower level from 10 d to 100 d. The expression level of PDS, ZDS and LCYB genes was gradually increased, and the expression level at 100 d closed to that of 18s rRNA. The expression levels of LCYE, VDE and ZEP genes were higher, showed no remarkable change over time, and the rank (from high to low) of expression level was LCYE, ZEP, and VDE.

In 7 different parts of lily, 12 key enzyme genes in carotenoid metabolism pathway were analyzed by semi-quantitative analysis: (1) 12 key enzyme genes in carotenoid metabolic pathway were all expressed in stigma, petal and stem; in root, PSY, LCYE and VDE genes were all not expressed, and CCS was expressed with extremely low level; in bulb, there was no expression except ZEP with weak level. (2) 12 key enzyme genes of carotenoid metabolism pathway were all expressed in stigma, anther, petal, leaf and stem, among them, the expression level of CCS was lower in 7 different parts of lily.

1.3 Cloning and expression analysis of LCYB

Using the leaves of Oriental lily after 30 days’ growth as experimental materials, the CDS of LCYB gene was cloned with the full-length of 1,503 bp, encoding 500 amino acids (Figure 4; Figure 5). Based on the analysis of NCBI CDD (Conserved domains database) (Figure 6), it was found that the amino acids of LCYB protein existed in the conserved regions of NADB_Rossmann superfamily, FixC superfamily and Carotene Cycl superfamily. Among them, the NADB_Rossmann superfamily had the longest conserved region with a NAD(P)H/NAD(P) (+) protein-binding domain folded by Rossmann, usually found in many metabolic pathways, for example, in dehydrogenase and oxidoreductase of glycolysis. The FixC superfamily was a kind of dehydrogenase (flavoprotein), which had the functions of energy production and transformation. The Carotene Cycl superfamily protein included β-lycopene and ε-cyclase, which formed β and ε carotene, respectively. Four amino acid fragments with the same domain were selected from the model plant Arabidopsis thaliana and sequenced by DNAMAN. The results showed that there was high sequence similarity in domains. And the Carotene Cycl superfamily was the representative family of LCYB gene (Figure 7). Analyzed by Protparam, the isoelectric point (pI) of LCYB protein was 7.60 and the instability coefficient was 38.46, which was less than the threshold value and was a stable protein. The total average hydrophilicity was -0.151, therefore, the LCYB protein was predicted to be hydrophilic protein.

1.4 LCYB phylogenetic analysis

In order to analyze the evolutionary relationship of lycopene β-cyclase (LCYB) gene between different species, plants with different relative branches were selected from Phytozome v12.1 database. BLASTP was used to carry out genome-wide search on the dicotyledonous plants such as Arabidopsis thaliana, Carica papaya, Populus trichocarpa, Fragaria vesca, Malus domestica, Vitis vinifera, Solanum lycopersicum, Amborella trichopoda, and Aquilegia coerulea, monocotyledonous plants, such as Musaacuminata, Zea mays, and Oryza sativa, Lycopodiophytes, for example, Selaginella moellendorffii, bryophytes, for example, Marchantia polymorpha, and Chlorophytes, such as Chlamydomonas reinhardtii, Dunaliella salina and Volvox carteri (Figure 8) (Du et al., 2015).

According to the alignment results of lily LCYB amino acid sequence in Phytozome v12.1 database, 50 amino acid sequences of other plants were selected to construct phylogenetic tree (Figure 9). The results showed that Malus domestica and Fragaria vesca of the rosaceae family, Selaginella moellendorffii of Lycopodiophyta and Marchantia polymorpha of bryophyta, and Chlamydomonas reinhardtii, Dunaliella salina and Volvox carteri of Chlorophyta, were drawn to the same branch and the support rate was higher than 50%. Lily was drawn to the same branch with Musaacuminata (both monocotyledonous plants). Although the support rate was low, to some extent, it reflected the closer relationship between lily and Musaacuminata compared with other plants.

2 Discussion

The content of carotenoid is significant different in different parts of lily. The total carotenoid content in the lily anther is significantly higher than that in other parts, which is related to vital functions of carotenoids in anther development. The anther wall consists of four parts: epidermal layer, fibrous layer, middle layer and tapetum. The tapetum is a special cell layer around anther. Its binuclear or polynuclear structure leads to more deoxynucleotides and proteins in the cells. In addition, there are nutrients such as grease and carotenoid, which can provide needed nutrients for pollen grain development (Gu, 2014).

Except for the low expression of ZEP gene, other key enzyme genes are not expressed in the lily bulb, and the total carotenoid content in the bulb is significantly lower than that in other parts. The main function of bulb is to store carbohydrates, and the metabolism of starch-sucrose plays a dominant role in the bulb. In the root of lily, all the key enzyme genes are expressed except PSY, LCYE and VDE, but the content of total carotenoid in root is still significantly lower than that in other parts. This is mainly due to the fact that PSY gene is the upstream key gene of carotenoid metabolism pathway (Kim et al., 2010; Huo et al., 2011; Zhang et al., 2014), and the loss of its expression results in low content of total carotenoid in the root. Except for root and bulb, LCYE and VDE gene are expressed in other parts, and the loss of LCYE and VDE gene expression is consistent with the loss of PSY gene expression, which indicates that LCYE and VDE gene expression are closely related to PSY gene expression. In the stem of lily, all the key enzyme genes are highly expressed, but the total carotenoid content is still relatively low, and there might be unknown regulatory genes in the metabolism pathway of carotenoid (Kishimoto et al., 2005). At different growth stages of lily leaves, the expression of key enzyme genes, PDS, ZDS and LCYB, gradually increases with the growth of lily leaves, and shows an obvious trend with time. Moreover, the increasing trend is consistent with that of carotenoid content (Furubayashi et al., 2014), which indicates that PDS, ZDS and LCYB play key roles in carotenoid metabolism pathway and show great significance in research.

The key branch point for lycopene β-cyclase (LCYB) to catalyze carotenoid metabolism pathway is the cyclization of lycopene (Yang et al., 2007; Li et al., 2010; Wu et al., 2011). The researches on LCYB gene provide a theoretical basis for further study on the metabolic pathway of carotenoid and the mechanism of carotenoid acting in color (Yamagishi et al., 2010). In this study, the identification, evolution and expression of LCYB gene were discussed in depth. The phylogenetic tree was constructed by comparing and analyzing the amino acid sequences of LCYB gene in the whole genome of known plants, which was helpful to further study the origin and evolution mechanism involved in LCYB gene and provide a scientific basis for studying the cellular function of LCYB gene.

3 Materials and Methods

3.1 Experimental materials

Oriental lily (Lilium ‘justina’) variety in Practice Base Greenhouse (Flower Bulb Greenhouse) of Beijing University of Agriculture was used as material for this experiment. The different parts of lily bulb growing for 100 days were collected in greenhouse, that was, style, anther, petal, leaf, stem, bulb, root, and leaves with different growth cycles (10, 25, 50, 75 and 100 days), which were put into liquid nitrogen quickly after sampling, and frozen at -80°C for later use.

3.2 Determination of carotenoid and chlorophyll content

The content of carotenoid and chlorophyll in lily was measured in this experiment by organic solvent method. Three samples were used as biological duplicates. After grinding, 0.250,0 g plant samples were accurately weighed, and 25 mL mixed liquor of acetone and anhydrous ethanol with the ratio of 1:1 was added into quickly. Then, they were mixed and kept in dark place for 4 hours. With the mixed liquor of acetone and anhydrous ethanol with the ratio of 1:1 as a blank control, D30 nucleic acid protein analyzer was used to measure the absorbance values of the wavelength of 470 nm, 474 nm, 485 nm, 642.5 nm, 649 nm, and 665 nm under A (absorbance value) procedure. The obtained data were sorted by Excel software and statistically analyzed by One-way ANOVA of SPSS17.0 software.

3.3 Semi-quantitative expression analysis of key enzyme genes in lily carotenoid metabolic pathway

Based on the results of constructed lily transcriptome library sequencing, specific primers of the key enzyme genes in carotenoid metabolic pathway were designed (Table 1). PSY, PDS, Z-ISO, ZDS, CRTISO, LCYB, LCYE, HYB, VDE, ZEP, CCS, and NCED were amplified and performed semi-quantitative expression analysis (Wang et al., 2015).

The total RNA of plant samples was extracted by EASYspinPlus plant RNA rapid extraction kit of Aidlab Biotechnologies Co., Ltd. cDNA template was obtained through RNA reverse transcription by TransScript First-Strand cDNA Synthesis SuperMix kit of TransGen Biotech Company. Using 18S rRNA of Lilium as reference gene, PCR with different cycle numbers of 20, 22, 24, 26, 28, 30, 32, 34, and 36 was performed. According to the electrophoretic results (Figure 10), the optimum cycle number of Lilium semi-quantitative RT-PCR was 32.

PCR system (25 μL): 1 μL cDNA template, 1 μL Primer 1 (10 μm), 1 μL Primer 2 (10 μm), 12.5 μL 2×Taq PCR Master Mix, and 9.5 μL ddH₂O. PCR program: pre denature at 94°C for 5 min; denature at 94°C for 40 s, anneal at 55°C for 30 s, extend at 72°C for 40 s, 32 cycles; extend at 72°C for 10 min; stay warm at 4°C; the relative expression of PCR products was analyzed by 1% agarose gel electrophoresis (Yao et al., 2015; Li, 2016).

3.4 ORF amplification and determination of LCYB gene

According to the unigene information of LCYB gene in oriental lily ‘Justina’ transcriptome sequencing results, the initial position of LCYB gene open reading frame (ORF) was found by comparing with GeneBank. Later, the specific upstream Primer LCYB-F and downstream Primer LCYB-R were designed based on the combination with unigene sequence. PCR program: pre denature at 94°C for 5 min; denature at 94°C for 45 s, anneal at 55°C for 30 s, extend at 72°C for 1 min, 32 cycles; extend at 72°C for 10 min; stay warm at 4°C. The final products of PCR were detected by agarose gel electrophoresis, and target fragments with gel extraction were recycled and used for objective cloning and sequencing.

3.5 LCYB bioinformatics analysis

After the ORF of LCYB was translated into protein by DNAStar, Blastp and InterProScan of NCBI (http://www.ebi.ac.uk/Tools/pfa/iprscan/) were used to analyze the conserved domain of LCYB gene coding protein sequence (Zhang et al., 2003). The physicochemical properties of protein were analyzed by ProtParam, the homologous sequence of LCYB was downloaded from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) database, and the phylogenetic tree was constructed by ML algorithm using Mega v5.0 software.

Authors’ contributions

LJ and WL were the designers and executors of this study; LJ finished data analysis and paper writing; DR participated in the experimental design and test result analysis; CJT and ZKZ were the designers and principals of the project, guiding the experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was co-funded by the Science and Technology Plan Item in 2015 of the Education Commission of Beijing (KM201510020011), the Beijing Laboratory Project of Urban and Rural Ecological Environment (PXM2019_014207_000012), the Collaborative Innovation Center for Beijing Forest and Fruit Industry Eco-environmental function Promotion (PXM2018_014207_000024) and Beijing Natural Science Foundation-Municipal Education Commission Co-funding Project (KZ2018100200-29).

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