www.nature.com/scientificreports OPEN Transcriptome profling of the fowering transition in safron (Crocus sativus L.) Jing Hu1, Yuping Liu1, Xiaohui Tang1, Huajing Rao1, Chaoxiang Ren1, Jiang Chen1, Qinghua Wu1, Yi Jiang2, Fuchang Geng3 & Jin Pei1 ✉ Safron, derived from the stigma of Crocus sativus, is not only a valuable traditional Chinese medicine but also the expensive spice and dye. Its yield and quality are seriously infuenced by its fowering transition. However, the molecular regulatory mechanism of the fowering transition in C. sativus is still unknown. In this study, we performed morphological, physiological and transcriptomic analyses using apical bud samples from C. sativus during the foral transition process. Morphological results indicated that the fowering transition process could be divided into three stages: an undiferentiated period, the early fower bud diferentiation period, and the late fower bud diferentiation period. Sugar, gibberellin (GA3), auxin (IAA) and zeatin (ZT) levels were steadily upregulated, while starch and abscisic acid (ABA) levels were gradually downregulated. Transcriptomic analysis showed that a total of 60 203 unigenes were identifed, among which 19 490 were signifcantly diferentially expressed. Of these, 165 unigenes were involved in fowering and were signifcantly enriched in the sugar metabolism, hormone signal transduction, cell cycle regulatory, photoperiod and autonomous pathways. Based on the above analysis, a hypothetical model for the regulatory networks of the safron fowering transition was proposed. This study lays a theoretical basis for the genetic regulation of fowering in C. sativus. Crocus sativus L., commonly called safron, is a perennial stemless herb belonging to the family Iridaceae (mono- cots), which is widely distributed in Iran, Spain, Greece, Italy and Nepal1. Due to the triploidy of its chromo- somes, this plant produces sterile fowers and reproduces asexually by corm nutrition. Safron was introduced to China from abroad, passing through Tibet, and has been successfully cultivated in many of its provinces, such as Shanghai, Zhejiang, Sichuan and Anhui, since the 1970s. Te fower, the most valuable part of safron, consists of six tepals, three stamens and three stigmas. Among these, the stigma is widely used as a spice or coloring and favoring agent in both the agro-food and cosmetic industries2. Te stigma is also used as a medicine due to its important pharmacological efciency3. Tus, safron is greatly required worldwide due to its wide use. However, in recent years, the safron fower has experienced increased incidences of withering, rotting, and delayed fower- ing, which has severely afected the quality and quantity of its stigmas and restricted the sustainable development of the safron industry. Terefore, this study on the molecular regulatory mechanisms of the safron fowering transition is particularly urgent and important for understanding and solving the problems related to safron fowering. Te complex process of the fowering transition is coregulated by both the external environment and the internal factors in plants to ensure fowering at an appropriate time4. In the model plant Arabidopsis thaliana, the flowering transition was found to mainly involve six regulatory pathways: the vernalization, photoper- iod, gibberellin, sugar metabolism, autonomous and age pathways5,6. Tese pathways converge to regulate the expression of fowering-related genes, such as FLOWER LOCUS T (FT), CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), LEAFY (LFY), APETALA1 (AP1), APETALA2 (AP2) and APETALA3 (AP3), which irreversibly induce the transition from the vegetative meristem to the foral meristem6,7. Te above pathways also play important roles in the regulation of fower induction in other plants. Research has shown that sugar, as both the energy substance and the signal molecule, positively mediates the fowering transition of grape8. IAA could accelerate the fowering process in Rosa chinensis, but ABA inhibits the process by interacting with 1State Key Laboratory of Traditional Chinese Medicine Resources Research and Development, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China. 2New Zealand Academy of Chinese Medicine Science, Christchurch, 8014, New Zealand. 3The Good Doctor Pharmaceutical group co. LTD, Mianyang, 622650, China. ✉e-mail: [email protected] SCIENTIFIC REPORTS | (2020) 10:9680 | https://doi.org/10.1038/s41598-020-66675-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Morphological characteristics of safron apical bud during foral transition process. (A,B) Shown the fower bud undiferentiated period (DS); (C,D) Shown the early fower bud diferentiation (BS); (E,F) Shown the late fower bud diferentiation (FS). sugar signals9. It has been extensively reported that vernalization regulates fowering in cereals10–12 and beets13, and a series of vernalization-related genes, including VRN1, VRN2, VIN1 and VIN2, have been identifed in wheat14. Rong Zhou et al.15 identifed and confrmed the sesame CO-like (COL) gene family from sesame genome data. Te CO gene encodes a B-box zinc-fnger transcription factor, which plays a central role in the photoperiod response and fowering regulation in Arabidopsis16. In addition, studies in other species, such as soybean, cotton, potato, and maize, also showed that the photoperiod pathway largely afects plant fowering17–20. Although a few studies have reported on safron EST, transcript data and functions of some genes21–27, most of which focused on apocarotenoid biosynthesis, corm sprouting and stigma development, little reported about the regulatory mechanisms of the fowering transition in safron. Tus, in this study, a transcriptomic analysis was performed by high-throughput sequencing. Te diferentially expressed genes (DEGs) related to the fowering transition during the three stages were analyzed. Additionally, the morphological changes in the safron fowering transition were observed, and changes in starch, soluble sugar and endogenous hormone contents were measured to comprehensively understand the regulatory networks of the safron fowering transition. Te results of this study lay a foundation for future studies and cultivation eforts, especially for the fowering of Crocus sativus L. Results Morphological characteristics of the safron fowering transition. Based on the morphological changes in the safron apical bud meristem from vegetative to reproductive growth, we divided the continuous growth process into three stages: fower bud undiferentiated period (DS), early fower bud diferentiation (BS), and late fower bud diferentiation (FS). In the undiferentiated period, the safron fower bud was small, less than or equal to 1 mm in length, and the apical growth point appeared semi-conical (Fig. 1A,B). Tis period was also regarded as the vegetative growth stage because the safron was gradually breaking dormancy and the foral primordium had not yet formed. At the early fower bud diferentiation stage, the length of the fower bud was approximately 1.5–2.0 mm, the growth point had been obviously raised and perianth primordia began to appear, indicating that the safron had trans- formed from vegetative to reproductive growth (Fig. 1C,D). In the late fower bud diferentiation stage, the fower bud was longer than 3 mm. Te diferentiation region of the inner bud had become wider and elongated, and the pistil primordia had begun to diferentiate (Fig. 1E,F). SCIENTIFIC REPORTS | (2020) 10:9680 | https://doi.org/10.1038/s41598-020-66675-6 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. Te starch and soluble sugar contents of apical buds during the fowering transition process in safron. (A) starch content and (B) soluble sugar content. Values are means of three replicates ± SE. Figure 3. Te hormone contents of apical buds during the fowering transition process in safron. (A) Abscisic acid (ABA); (B) Gibberellin acid 3 (GA3); (C) Auxin (IAA); (D) zeatin (ZT). Sugar and hormone contents during the fowering transition process Te levels of starch and soluble sugar in the apical buds were measured at three stages during the fowering tran- sition (Fig. 2). In the safron apical buds, the starch content was high in DS, slowly decreased by 11.33% from DS to BS and sharply decreased by 36.41% between BS and FS (Fig. 2A). In contrast, the soluble sugar content continuously increased by 65.14% from DS to FS (Fig. 2B). Te hormone contents were also analyzed in the apical buds at three stages during the fowering transition process (Fig. 3). Te ABA content increased by 15.91% between DS and BS but sharply decreased by 48.53% from BS to FS (Fig. 3A). Te GA3 content increased by 86.69% from DS to BS and slowly decreased by 8.65% between BS and FS (Fig. 3B). Te IAA content continuously increased by 80.96% from DS to FS (Fig. 3C). In addition, the ZT content showed low levels between DS and BS but then sharply increased by 98.11% from BS to FS (Fig. 3D). General analysis of safron transcriptome data. In this study, a total of 430 853 316 raw reads were obtained from the three stages of safron buds based on three biological duplications for each stage. Afer the elimination of low-quality reads and adaptor sequences, 422 584 176 clean reads were selected for further anal- ysis (Table 1). Finally, 60 203 unigenes with a mean size of 1045 bp were assembled with lengths ranging from 201 to 14 704 bp (Fig. S1 of Appendix S1). Te GC content and N50 of the unigenes were 43.49% and 1532 bp, respectively, indicating high assembly quality. Among the 60 203 unigenes, 34 144 (56.71%), 23 618 (39.23%), 21 481 (35.68%), and 14 671 (24.37%) unigenes were successfully annotated according to the NR, Swiss-Prot, KOG and KEGG databases, respectively (Fig. 4A). Based on the NR database, 19.57% of the unigenes showed homology (1e−20 < E-value <1e–5), 44.06% of those showed strong homology (1e−100 < E-value <1e−20) and the remaining 36.37% showed very strong homology (E-value <1e − 100) to the available plant sequences (Fig. 4B). As shown in Fig.