Lilium spp., one of the world’s famous ornamental flowers, is mainly cultivated as cut flower, whose ornamental value lies in the floral organ. It usually comes in colors of red, orange, white and pink with the absence of blue or purple. Lilium cernum., also known as Lilium pinifolium, falls into the Sect. Sinomartagon of Lilium genus and is also one of the most important wild species in Asiatic hybrids. Among such distribution areas as Northeast China (Jilin Province and Liaoning Province), the Korean Peninsula and Russia, the Changbai Mountains are the major one of Lilium cernum., the only wild fragrant purplish-red Lilium spp. in the Northeast China. Plants on the Changbai Mountain (the Baekdu Mountain in North Korea) have been in the cycle of glaciation and interglaciation due to height change of the mountain, resulting in that a series of low and medium density populations have been subjected to extensive genetic variation (Chung et al., 2014). The case is same for the wild Lilium cernum species, which are under different color variations.

The main reason causing color variations rests on the different types and contents of anthocyanidins, wherein the carotenoids, flavonoids and alkaloids are the major pigments contributing to color formation of flowers. Purple, crimson, pink and other colors of Lilium spp. are generally caused by different contents of flavonoid (anthocyanin), for example the ‘Holean’, ‘Monte Negro’ and ‘Red Carrpet’ of purplish-red Lilium Asiatic., while the white Lilium spp. does not contain any anthocyanin or flavonol (Yamagishi et al., 2012; Nørbæk and Kondo, 1999). Many studies have pointed out that the biosynthesis of anthocyanins in flowers are primarily regulated at the transcriptional level of biosynthesis genes, which involves multiple transcription factors. LhR3MYB1 and LhR3MYB2 in Lilium spp are capable of inhibiting the accumulation of anthocyanin while the latter is more intensely involved in the negative regulation to inhibit the same (Sakai et al., 2019). CmMYB4 and CmMYB5 in Chrysanthemum×Morifolium negatively regulate the biosynthesis of its anthocyanin (Hong et al., 2019) and additionally, the MYB6 in Populus tomentosa (Wang et al., 2019) and VvMYBA1 and VvMYBA1-1in Vitis vinifera (Xu et al., 2019) are conducive to the biosynthesis of their anthocyanins and proanthocyanidins. In addition, the application of the exogenous ABA can increase the up-regulated expression of MdMYB110ac in Malus pumila. and further increase the content of anthocyanin in the pericarp of Malus pumila (Cho et al., 2020), which demonstrate that the MYB gene family and various subfamilies are conducive to the positive and negative regulations on anthocyanin synthesis.

However, MYB gene alone cannot regulate the anthocyanin synthesis, the bHLH and WD40 are also required to form MBW compound so as to activate the promoter of anthocyanin synthesis structure. The FhTTG1 gene, which can highly activate the gene promoter relating to biosynthesis of anthocyanin (Shan et al., 2019), in the WD40 family of Freesia hybrida is expressed in parallel with the accumulation of proanthocyanidin and anthocyanin in Freesia hybrida. 138 transcription factors bHLH are screened out from the genome of Ziziphus jujube, wherein the ZjTT8, ZjGL3a and ZjGL3b of the III subfamily are highly correlated with the anthocyanin synthesis (Shi et al., 2019).

The research group discovered in the field survey that the wild Lilium cernum experienced color variation, with colors ranging from dark purple to white. The shades and coloration area proportions vary in individuals (Figure 1) and pink ones (Figure 1B) are common while the white (Figure 1A), purple (Figure 1D) and even pinkish white ones (Figure 1C) quite rare. Some studies believe that the pink Lilium Asiatic may be generated from the Lilium cernum (Yamagishi et al., 2014). Furthermore, recent reports suggest that the Japanese scholars divide the Lilium cernum into 2 categories based on the flower color, namely the ordinary pink ones and the variable white ones (L.cernuum var.album) (Yamagishi, 2020), which are consistent with the varieties discovered by the research group in the field (Figure 1A). In addition, the research group also discovered the Purplish-red ones and the pinkish white ones apart from the two categories mentioned above. The purple one has always been a rare one of all the species of Lilium spp., which is also a variation seldomly observed in the wild Lilium spp. Moreover, the purplish-red one and the pinkish white one is completely the same in the early stage. Therefore, the change of color of Lilium cernum from white to dark purple provides an opportunity for the research on the molecular regulation mechanism of anthocyanin biosynthesis of Lilium spp. and is of vital significance to the cultivation of new species of Lilium spp.

1 Results and Analysis

1.1 Quality of sequencing data

The transcriptome data obtained were assembled (Table 1), from which it could be seen that the number of transcripts was 118 091 with length of 992.93 bp on average. After screening and assembly, 57 521 Unigenes were obtained and the length of N50 was 1 227 bp (870.01 bp on average), ranging from 300 to 1 000 bp mostly. The obtained genes accounted for 73.29% of the total amount of Unigenes, indicating a high assembly integrity.

1.2 Unigene function annotation

25 678 annotated Unigene were obtained eventually after selecting the BLAST parameter with E-value not greater than 1e^-5 and HMMER parameter with E-value not greater than 1e^-10 (Table 2), among which 12 531 annotated ones were with lengths ranging from 300 to1 000 bp and 13 147 annotated ones were above 1 000 bp in length.

1.3 Differently expressed genes

The number of differential genes of different samples were quite different. In Group Bud Stage versus Purplish-red, the number of differential genes was the largest (up to 3 665) and that of the up-regulated genes was also the largest (up to 2 746). In Group Purplish-red versus Pinkish-white, the number of down-regulated genes was larger than that of the up-regulated genes, while differentially expressed gene sets of other comparison groups indicated that the number of up-regulated genes was larger than that of down-regulated genes. However, in Group Color Conversion Stage versus Pinkish-white, there were only 23 differential genes, wherein the number of up-regulated genes was 3 and that of the down-regulated genes 20 (Table 3).

1.4 Annotation of KEGG differentially expressed genes

1.4.1 Annotation of differential genes in the metabolic pathway of anthocyanin

6 structural genes differentially expressed in the synthetic pathway of anthocyanin were screened out via the screening of differential genes in metabolic pathway of anthocyanin during different flower development processes of Lilium cernum with specific names, expressions and function annotations of the genes (Table 4). In Group Bud Stage versus Color Conversion Stage during the early flower development, the expressions of DFR, CHS, ANR and ANS were all up-regulated while the expression of FOMT was down-regulated (Table 5). As the flower development continued (in Group Color Conversion vs Purplish-red), the expression of ANS was up-regulated and that of agmatine coumaroyl transferase (ACT) down-regulated, while the expressions of other genes remained the same. In Group Purplish-red versus Pinkish-white, the expression of CHS was down-regulated and that of ACT up-regulated; in Group Bud Stage versus Purplish-red, the expressions of DFR and ANS were up-regulated and the expression of FOMT was down-regulated; in Group Bud Stage versus Pinkish-white, the expressions of 6 differential genes are as follows: the expressions of DFR, CHS, ANR and ANS were up-regulated and the expression of FOMT was down-regulated. Nevertheless, in the Group Color Conversion versus Pinkish-white, the 6 differential genes in metabolic pathway of anthocyanin remained the same.

1.4.2 Annotation of differential genes (Color Conversion Stage and Pinkish-White)

In terms of two different colors of Lilium cernum., samples at color conversion stage and the pinkish-white state were quite similar in color but different in blossoming state. In the course of sequencing comparison, there were only 23 differentially expressed genes, which were annotated then. Among the 23 differentially expressed genes related to the metabolic process and energy synthesis, there were 5 genes related to cell control and movement. The results of functional annotation showed that (Table 5) 3 out of the 5 differentially expressed genes were from the MCO family and all associated with the synthesis and decomposition of lignin; While the wood cellulose synthase was related to the biosynthetic process and cell movement of cellulose. The expressions of such 5 genes were down-regulated in the process of transformation from the color conversion stage to the pinkish-white state.

1.5 Transcription factors regulation

1.5.1 MYB transcription factors

There were 2 125 transcription factors in the transcriptome database, wherein 244 were MYB and MYB family transcription factors. The MYB and its family members subjected to expression change in the blossoming process of Lilium cernum (Table 6). In the process of transformation from bud stage to color conversion stage, there were 15 genes including MYB2 with expressions up-regulated and 6 genes including MYB52 with expressions down-regulated and the rest 222 remained the same. Additionally, in the process of transformation from color conversion stage to purplish-red state, 7 genes including MYB98 were up-regulated, 3 genes including MYB73 were down-regulated and the rest 234 remained the same. Among the differential genes in Group Purplish-red versus Pinkish-white, 4 including MYB58 were up-regulated and 8 including MYB14 down-regulated, while the rest 232 remained the same. In Group Bud Stage versus Purplish-red, 3 genes including MYB98 were up-regulated and 3 genes including MYB protein L subtype×1 down-regulated, while the rest 238 remained the same. In Group Bud Stage versus Pinkish-white, 1 gene including MYB118 was up-regulated and 2 genes including MYB56 and MYB protein 3R-1 subtype×2 were down-regulated, while the rest 241 remained the same. In Group Color Conversion Stage versus Pinkish-white, expressions of 244 genes remained the same.

1.5.2 Transcription factors bHLH

There were 103 bHLH and bHLH family transcription factors, the bHLH and its family members subjected to expression change in the blossoming process of Lilium cernum (Table 7). In Group Bud Stage versus Color Conversion Stage, 2 genes including bHLH54 and bHLH-MYC were up-regulated and 3 genes including bHLH82 down-regulated, while the rest 98 remained the same. In Group Color Conversion State versus Purplish-red, 5 genes including bHLH30 were up-regulated and 2 genes including bHLH35 and bHLH52 down-regulated, while the rest 96 remained the same. In Group Purplish-red versus Pinkish-white, 3 genes including bHLH35 were up-regulated and 3 genes including bHLH30 down-regulated, while the rest 97 remained the same. In Group Bud Stage versus Purplish-red, 6 genes including bHLH30 were up-regulated and 4 genes including bHLH35 down-regulated, while the rest 93 remained the same. In Group Bud Stage versus Pinkish-white, 2 genes including bHLH54 and bHLH-MYC were up-regulated and 3 genes including bHLH52down-regulated, while the rest 98 remained the same. However, in Group Color Conversion Stage versus Pinkish-white, expressions of 103 genes remained the same.

1.6 qRT-PCR verification of differentially expressed genes

6 differentially expressed genes were screened out for qRT-PCR verification (Table 8). The results showed that the expression of CHS is the highest at color conversion stage and the second highest at the pinkish-white state, between which the difference was not significant. However, the expression of CHS was the lowest at bud stage, showing an extremely significant difference in comparison with that at color conversion stage, purplish-red state and pinkish-white state; the expression of ANS increased constantly in the process of transformation from bud stage to purplish-red state and there was a significant difference between expressions at color conversion stage and pinkish-white state; the expression of MYB98 kept increasing in the process of transformation from bud stage to purplish-red state, while the difference among expressions at pinkish-white state, bud stage and color conversion stage was not significant, but the difference between expressions at pinkish-white state and the purplish-red state was extremely significant; the expression of MYB14 was not significantly different at bud stage, color conversion stage or pinkish-white state while that at the purplish-red state was the highest, showing an extremely significant difference in comparison with other stages or state; the expression of bHLH52 was the highest at bud stage and the lowest at purplish-red state, while the difference between expressions at color conversion stage and pinkish-white state was not significant; the expression of bHLH95 at purplish-red state was the highest and showed an extremely significant difference in comparison with that at bud stage, color conversion stage and pinkish-white state.

2 Discussions

Based on filed observation, the research group divided the blossoming process of Lilium cernum. into two primary stages: the pre-color conversion stage, which included the bud stage and color conversion stage, where there was no obvious change in flower color and the bud kept growing; the post-color conversion stage, at which the floral organs of Lilium cernum. developed into such two states as purplish-red state and the pinkish-white state and the colors kept or stopped being darkened. Therefore, discussions were carried out on the basis of two stages and two color states.

In the research of flowering mechanism, many researches suggested that the color change was mainly caused by the differences in types and contents of anthocyanin and anthocyanidin, while genes involved in the anthocyanin synthesis could be divided into two major categories, namely the structural genes of anthocyanin synthesis, such as GSH and ANS, and the transcription factors activating or inhibiting the enzyme expression. 6 structural genes were screened out after transcriptome sequencing based on the different development states and states of the floral organs of Lilium cernum. and comparing the difference in expression of structural genes of anthocyanin synthesis of floral organs at different stages, and they were: DFR, CHS, ANR, ANS, FOMT and ACT. Wherein, the DFR is able to catalyze the colorless anthocyanin products into colorless pelargonidin, colorless cyanidin and colorless delphinidin; CHI is the first important structural enzyme in the pathway of anthocyanin synthesis, which can catalyze the 4-coumaroyl-CoA into CHI, and CHI is then transformed into colorless anthocyanin by a series of catalytic reactions; ANS then catalyze the colorless anthocyanidin into anthocyanin. This process is also obtained from such flowers as Osmanthus fragrans (Ma et al., 2017) and Brunfelsia acuminata (Min et al., 2016). However, the color change was not obvious in the pre-color conversion stage of Lilium cernum. with a little appearance of purple. In this process, the ANR and FOMT were both up-regulated, which might inhibit or restore the pigments subjected to color conversion. It was pointed out in researches of regulation on colors of Malus crabapple (Li et al., 2019) and Camellia sinensis (Zhao et al., 2017) that the function of ANR was opposite to that of ANS, while ANR was responsible for reducing the anthocyanin synthesis; FOMT was a member of the methyltransferase family of transferases, which regulated the color response of plant tissues by changing the types, chemical stability, water solubility and other characters of anthocyanin. The purple trait in petals of Paeonia spp. were regulated by the methylation of anthocyanin (Du et al., 2015). In lilium cernum colors might be regulated through a similar pathway since DFR, CHS and ANS were up-regulated to increase anthocyanidin at the pre-color conversion stage and ANR and FOMT are up-regulated to reduce the synthesized anthocyanidin, while the same differential expression of genes also occurred in the process of development of Lilium cernum from the bud stage to the white state. Meanwhile, CHS and ANS were verified by qRT-PCR and it was also indicated that the expressions of CHS and ANS increased at the pre-color conversion stage, which was consistent with the sequencing results.

Additionally, the colors of Lilium cernum at color conversion stage and pinkish-white state were basically the same, while the blossoming states were different. After comparing the differentially expressed genes at this stage and state, there were only 23 differentially expressed genes, among which 5 were related to cell movement and cellulose synthesis. Meanwhile, after comparing the expressions of DFR, CHS, ANR, ANS, FOMT and ACT in the pathways of anthocyanin synthesis at color conversion stage and pinkish-white state, it was discovered that the expressions of said 6 genes remained the same. According to comparison by qRT-PCR between the expressions of CHS and ANS at color conversion stage and pinkish-white state, the expressions of CHS at color conversion stage and pinkish-white state was not significantly different, and that of ANS was not extremely different neither, which were basically consistent with the sequencing results. It was proved that the anthocyanin synthesis processes at color conversion stage and pinkish-white state were consistent but the blossoming states were different. As a result, it was speculated that the blossoming process of pinkish-white flowers might be advanced at the color conversion stage due to internal and external environmental changes or other reasons, resulting in blossoming before the completion of color conversion. Nevertheless, since there are few researches on morphological changes during the blossoming process and some scholars point out that the curvature or wrinkle of petals is caused by uneven expansion of the cells in petals (Kawabata et al., 2011), the internal mechanism of the uneven distribution of anthocyanidins on the petals of Lilium cernum.caused by advanced blossoming needs to be supported by more experiments.

According to many researches and reports, the MYB, bHLH and WD40 transcription factors played a significant role in flower color regulation. A large number of MYB and bHLH transcription factors were found in the blossoming process of Lilium cernum. and the MYB and bHLH transcription factors differed in the type and amount of differential expression at different development stages of flowers. Based on the sequencing results, the differentially expressed MYB and bHLH transcription factors were mostly concentrated in the whole process transformation from bud stage to purplish-red state, but less concentrated in the process of transformation from bud stage to pinkish-white state. The MYB and bHLH transcription factors of Lilium cernum. were related to the change of flower colors. For instance, the MYB14, MYB98, MYBC1, PHL7, PHL8, cytochrome P45094A1, bHLH30 and bHLH51 in the process of transformation from color conversion stage to purplish-red state (from light to dark) were all up-regulated, while such same transcription factors in the process of transformation from purplish-red state to pinkish-white state (from dark to light) were down-regulated. The same phenomenon also occurred to transcription factors MYB13, bHLH52 and bHLH35. Such four transcription factors as MYB98, MYB14, bHLH52 and bHLH95 were selected for qRT-PCR verification and the results also indicated that the expressions of MYB98 and MYB14increased in the process of transformation from color conversion stage to purplish-red state the difference was extremely significant, and that compared with the pinkish-white state, the expressions of MYB98 and MYB14 at purplish-red state decreased and the difference was extremely significant. The expressions of bHLH52 and bHLH95 were also consistent with the sequencing results. These phenomena indicated that such transcription factors as MYB14, MYB98, MYBC1, PHL7, PHL8, cytochrome P45094A1, bHLH30, bHLH51, MYB13,bHLH52 and bHLH35 played important positive and negative regulatory roles in the process of anthocyanin synthesis and accumulation. In addition, many researches also pointed out that MYB, bHLH and WD40 interacted with each other to form a binary or ternary complex for the joint regulation on structural genes and anthocyanin metabolic synthesis. However, the differentially expressed transcription factor WD40 was not discovered in this research, which may be that the anthocyanin of Lilium cernum. was regulated by the binary complex of bHLH and R2R3-MYB instead of the ternary complex (Sakai et al., 2011).

Unfortunately, the whole genome sequencing of Lilium spp. has not been completed yet. The MYB and bHLH transcription factor families are considerably large and there are many differences among transcription factors of different species of Lilium spp.Furthermore, no research of the color regulation transcription factors of Lilium spp. has been conducted with respect to the types mentioned above at home and abroad, consequently, specific functions of these transcription factors need to be further verified by more experiments. 

3 Materials and Methods

3.1 Sampling

The well-grown Lilium cernum planted in the horticultural farm of Jilin Agricultural Science and Technology University were selected as the experimental materials. Seeds of the Lilium cernum were from Huashan Village, Linjiang City, Jiling Province (N41.881277, E126.829992 and 567.5m above sea level) and floral organs were collected on June 23^rd, 2019. With respect to the four colors of Liliun cernum (Figure 1), two more obvious variations (Figure 1D (Purplish-red) and Figure 1C (Pinkish-white)) were selected for sampling since there was only 1 white Liliun cernum with 2 flowers only, which were unable to meet the demands of subsequent experiments. Meanwhile, the development processes of Type C and Type D Liliun cernum before the H stage were identical and their colors began to change after the color conversion stage. Therefore, 2 flower development processes (Figure 1K (Bud Stage) and Figure 1H (Color Conversion Stage)) were selected for sampling. Afterwards, samples were put in pre-sterilized sterile bags. Three parallel samplings were counted as three repetitions. Stamens and pistils were removed after samples in each group were marked. The petals were immediately frozen with liquid nitrogen and brought back to the lab for storage in refrigerator at -80°C.

3.2 RNA extraction

Total RNA from the petals of Lilium cernum. was extracted by using the polysaccharide and polyphenol total RNA isolation kit (TIANGEN, CHINA). After the total RNA was tested and qualified by agarose gel electrophoresis, the OD value was measured with the microvolume UV-Vis spectrophotometer (NanoPhotometerTM P-Class, USA). A260/280 ranged from 1.70 to 1.90, and the DNA concentration was above 100 μg·L^-1. The extraction was stored in refrigerator at -80℃ after satisfying demands of subsequent experiments.

3.3 Database establishment and sequencing

Transcriptome sequencing was performed on the total RNA of the extracted petals by Beijing Biomarker Technologies Co., Ltd. with the adoption of Illumina novaseq 6000 (Illumina, The United States). In order to ensure the data quality, the raw data was filtered and Trinity (v2.4.0) was used for data assembly. After the assembly, BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was applied to compare the gene annotations with such databases as NR (ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nr.gz), Swiss-Prot (ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz), GO (http://geneontology.org), COG (https://www.ncbi.nlm.nih.gov/COG/), KOG (ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kyva), eggNOG4.5 (http://eggnogdb.embl.de/) and KEGG (http://www.kegg.jp/kegg). Afterwards, DESeq2 (v1.23.10) was used to analyze the differential expressions between sample groups, and the differentially expressed genes were classified into COG and eggNOG respectively. After comparing with the KEGG database, the differential genes in the anthocyanin metabolic pathway were screened out.

3.4 Screening of differentially expressed genes

DESeq2 (v1.25.9) was applied to the analysis of differential expressions between sample groups so as to obtain differentially expressed gene sets between different samples. Among which, FC (Fold Change) ≥ 2 was marked as up-regulated genes and FC<0.05 was marked as down-regulated genes, which was of statistical significance (P<0.05).

3.5 Determination by qRT-PCR

Quantitative real-time PCR (qRT-PCR) was used to determine the transcript level of differentially expressed genes in the four developmental processes of Lilium spp. SYBR Premix Ex Tag (Tli RnaseH Plus, TaKaRa) was used for qRT-PCR while cDNA (diluted to 20×) served as the template, and the operation was carried out according to the instructions. The gene-specific primers for qRT-PCR were designed by Primer Express (v3.0), and the reference gene adopted the general actin of Lilium spp. (LhActin, NCBI accession number: JX826390). qRT-PCR specific primers and endogenous reference were listed (Table 9).

Authors’ contributions

CSP and ZQQ designed and executed the experiments of this research; WW collected the samples and LSH performed the data analysis; LRR participated in experiment design and experiment result analysis; YTY wrote and revised the paper. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the “Thirteenth Five-Year” Science and Technology Project of the Education Department of Jilin Province (JJKH20190982KJ) and the Central Finance Forestry Science and Technology Promotion Demonstration Project (JLT2020-39).

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PMid:29244739 PMCid:PMC6149802