Brassinosteroids (BRs), a kind of sterols that were first discovered and isolated from Brassica napus pollen, have been reported to be involved in many aspects of plant growth and development (Grove et al., 1979; Clouse, 1997). In the growth and development of plant roots, BRs were reported to participate in maintenance of meristem size, lateral root initiation, nodulation in legume species and root hair formation (Bao et al., 2004; Terakado et al., 2005; Hacham et al., 2011; Cheng et al., 2014). Moreover, the roots of BR deficient mutant were found significantly shortened (Müssig et al., 2003). In Arabidopsis, BR was found working cooperatively with auxin in promoting petiole elongation to response the shade stimulus (Kozuka et al., 2010). BRs were also reported to be involved in stem elongation, cell division, vascular differentiation, and leaf bending and epinasty (Thompson et al., 1982; Clouse et al., 1996; Tong and Chu, 2012; Hao et al., 2013). In field production, BRs are believed to improve crop yield and plant tolerance against environmental stress (Divi and Krishna, 2009).

To date, more than 70 kinds of BRs have been identified (Miransari, 2014). Among them, brassinolide (BL) is recognized as the most active form (Fujioka and Sakurai, 1997). With the rapid development of biotechnology, the biosynthesis and signaling pathways of this hormone have been well studied. BL is produced via the early C-6 oxidation pathway and late C-6 oxidation pathway (Noguchi, 2000). Numerous studies have shown that regulation of genes involved in BR biosynthesis pathway can result in alteration in BR accumulation and plant growth (Yoshimitsu et al., 2011; Zhiponova et al., 2013). Some genes, such as DWARF7 (DWF7), DWARF1 (DWF1), DE ETIOLATED2 (DET2), DWARF4 (DWF4), CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM (CPD), CYP85A2, and CYP90C1 have been verified to play roles in BR biosynthesis (Choe et al., 1998, 1999; Noguchi, 1999; Du and Poovaiah, 2005; Ohnishi et al., 2006, 2012; Nole-Wilson et al., 2010). Among them, DWF4 is reported to be a key gene in BR biosynthesis, which may control the putative rate-limiting step in the BR biosynthetic pathway (Choe et al., 1998, 2001).

The homeostasis of BRs is regulated by biosynthesis genes and catabolism genes (Tanaka and Okamoto, 2005). In Arabidopsis, BAS1/CYP734A1 is a major BR inactivating gene (Neff et al., 1996). In many higher plants, the CYP734A paralogs was also found (Thornton et al., 2011). In addition, genes involved in the signaling pathway of BRs have been well investigated in Arabidopsis. Brassinosteroid insensitive 1 (BRI1), BRI1-associated receptor kinase1 (BAK1), BR-signaling kinase1 (BSK1), BRI1 suppressor 1 (BSU1), bridging integrator 2 (BIN2), brassinazole resistant1 (BZR1), and BRI1-EMS-suppressor1 (BES1) were involved in the signaling pathway (Wang and He, 2004; Sun et al., 2010; Yan et al., 2012; Shi et al., 2013a,b; Zhang et al., 2014; Jiang et al., 2015). Among these genes, BRI1 works as the receptor for BRs, BZR1and BES1 are the transcription factors that regulate the growth of plants (Gallegobartolomé et al., 2012; Guo et al., 2013).

Numerous studies have been conducted to determine the function of BRs in plant growth. However, BR accumulation and its potential roles during carrot growth and development remain elusive. Carrot (Daucus carota L.) is a nutrient-rich root crop from the Apiaceae family (Luby et al., 2014; Xu et al., 2014; Wang et al., 2015). It is one of the most economically important members of Apiaceae plants, and the cultivated area of carrot is progressively increasing worldwide (Cavagnaro et al., 2011).

In this study, BR accumulation at five successive growth stages was examined in carrot. Expression profiles of genes involved in BR biosynthesis and signaling pathways were also analyzed. Besides, exogenous 24-epibrassinolide (24-EBL) was applied to carrot plants to study the putative effects of BR on carrot. The results of our work would shed novel insights into studies focusing on hormonal control of plant growth.

Phytohormones are very important factors affecting almost all the processes of plant growth (Singh and Savaldigoldstein, 2015). Brassinosteroids are a class of phytohormones with various functions in plants. They have been reported to play important roles in many developmental processes including primary root extension and lateral root formation, seed germination, cell and stem elongation, abiotic stress responses (Roddick and Guan, 1991; Clouse et al., 1996; Li and Chory, 1997; Bao et al., 2004; Sharma et al., 2015). Functions of BRs have been documented in many plants, such as, tomato, cucumber, rice, and maize (Montoya et al., 2005; Fu et al., 2008; Kir et al., 2015). However, relevant study regarding BRs in carrot is rare.

The precise role of a specific hormone on plant growth and development is not only a result of its available levels, but also relates to the plant species, and the target organ (Singh and Savaldigoldstein, 2015). In addition, the roles of a specific hormone also depend on the presence of other phytohormone and plant growth regulators (Nemhauser et al., 2006). BRs, acting as a growth-promoting hormone were reported to be most actively biosynthesized in actively growing tissues and function at the sites of synthesis. In Arabidopsis, the highest and the second highest level of endogenous BRs were observed in apical shoots and developing siliques which were actively developing organs, respectively (Shimada et al., 2003; Montoya et al., 2005).

In carrots, the content of bioactive BR levels at different stages and in different organs keeps changing (Figure 2). These results suggested that the roles of BRs were different at different developing stages. At stage1, the main function of BRs may be promoting the formation of roots. At stages 3 and 4, BRs may mainly promote the development of leaf blades and petioles, respectively. Here, the content of bioactive BRs was detected by ELISA. ELISA is an indirect approach for measuring BRs. According to previous reports, high performance liquid chromatography (HPLC), high performance liquid chromatography-mass spectrometry (HPLC-MS), and ELISA were compared in measuring BRs and they determined that ELISA can facilitate the determination of brassinosteroid phytohormones in plant (Pradko et al., 2014). Besides, based on the simple, rapid, fidelity and cost-effective ELISA method, many endogenous hormones in higher plant were detected, such as IAA, gibberellin (GA), and methyl jasmonate (MeJA) (Chen et al., 2009; Wang et al., 2015, 2016; Wu et al., 2016).

According to previous studies, BR plays important role in leaf growth and development. The well-known example is that exogenous BR can affect the bending of the lamina joint of rice (Mori et al., 2002). In rice, Dwarf1 gene was found to encode GTP-binding protein. The phenotype of its recessive mutant is dwarf (Li and Chory, 1997; Ross et al., 1997; Azpiroz et al., 1998). According to previous reports, the dwarf phenotype of dwarf mutants was associated with brassinosteroids and gibberellins (Hong et al., 2002). BR-deficient dwarf 1(brd1) mutant is a BR-related dwarf plant and the elongation of stem and leaves of which were reported to be repressed in rice (Hong et al., 2002). By contrast, exogenous BL stimulated the unrolling of leaf blades in wheat (Wada et al., 2014). In Arabidopsis, BR-deficient mutants appeared to have stunted shoot and small leaves (Nakaya et al., 2002). Here, the expression levels of genes involved in BR biosynthesis in leaf blades were higher than that in the root at the last three stages. The catabolism gene DcCYP734A1 has the same expression trend in leaf blades of carrot plants. In addition, the fresh weight of aerial part of treated carrot plants (0.5 mg/L) was significantly higher than that of control. After treating by 24-EBL, genes DcDWF4, DcCPD, and DcCYP85A2 in BRs biosynthesis pathway were down-regulated and catabolism gene DcCYP734A1 was up-regulated at the concentration of 0.5 mg/L in the leaf blades. These results suggested that BRs may play important roles in carrot leaf blade growth.

BRs regulate plant growth and development through promoting cell elongation and division (Fridman and Savaldi-Goldstein, 2013). In Arabidopsis, the size and intercellular spaces of matured leaves of deetiolated2 (det2) and dwarf1 (dwf1) mutants were smaller than those of wild plants. These phenotypes of det2 and dwf1 mutants were demonstrated to be the result of inhibited elongation and division of cells in Arabidopsis (Nakaya et al., 2002). Similar results were also observed in rice (Hong et al., 2002). Plant height is an important index for plant growth. BR was reported to be a crucial factor for plant height (Hong et al., 2002). In soybean, GmBRI1b (Glyma04g39160) was a putative BR receptor and was found high similar to those of AtBRI1 and pea PsBRI1. The petioles of GmBRI1b over-expression lines were longer than that of wild plants (Peng et al., 2016). Many BR-deficient mutants, such as dwarf4 (dwf4), and dwf7-1 in Arabidopsis, dwarf1-1 in sorghum, exhibited dwarf phenotypes due to deficiency in BR biosynthesis (Choe and Feldmann, 1998; Cheon et al., 2010; Fridman and Savaldi-Goldstein, 2013; Petti et al., 2015). Here, the biosynthesis genes and catabolism genes showed higher expression levels at the first two stages which suggested that BRs may be involved in actively growing of petioles. In petioles of carrot plants, after being treated by exogenous BL, expression levels of most biosynthesis genes were inhibited. However, the catabolism gene DcCYP734A1 was significantly up-regulated treated by exogenous BL at the concentration of 0.5 mg/L 24-EBL. Most signal transduction genes were also up-regulated when treated by 0.5 mg/L 24-EBL. Carrot plants treated by 24-EBL had increased height and aerial part weight. Meanwhile, the number of petioles in carrot plants increased after application of exogenous 24-EBL. All the above results suggested that application of exogenous 24-EBL promoted the growth of carrot petioles.

Carrot is a kind of root vegetable crop. The root of carrot is a good source of phytochemicals, such as carotenoids, anthocyanins, and other phenolic compounds. Till now, the brassinosteroid’s roles involved in carrot root growth and development are not clear. In Arabidopsis, application of exogenous brassinolide was reported to promote root elongation and lateral root germination (Gonzálezgarcía et al., 2011; Singh and Savaldigoldstein, 2015). However, BRs plays different roles in regulating nodule formation in different plant species. BRs can actively regulate the nodule numbers in pea, but function as an inhibitor in soybean nodule formation (Ferguson et al., 2005; Terakado et al., 2005). During the development of carrot, all biosynthesis and catabolism genes showed higher expression levels at the first two stages in root which suggested that BRs may be necessary for the early stage of carrot root growth and development. However, the expression levels of signal transduction genes do not show similar trend. After treatment, all the biosynthesis, catabolism and signal transduction genes were up-regulated. However, the root length and diameter of carrot plants treated with exogenous 24-EBL did not show significant changes in our study. Symons and his colleagues demonstrated that the long-distance transport of BRs cannot be detected in pea (Symons and Reid, 2004). In tomato, the long-distance transport of BRs is also lacking (Montoya et al., 2005). Moreover, BRs were thought to regulate root meristem size maintenance depending on their action site (Wei and Li, 2016). In Arabidopsis, the meristem size of root was reported to be controlled by BR signaling in the root epidermis (Gonzálezgarcía et al., 2011). In high plant, the extensive interactions were existed among different hormonal signaling pathways. Many kinds of hormones were involved in the regulation on plant growth and development (Nemhauser et al., 2006). In rice, the signaling pathways of auxin, ABA and GA3 controlling cell elongation are affected by other pathway involving brassinosteroids (Ephritikhine et al., 1999). In Arabidopsis, the growth of hypocotyls was reported to be controlled by auxin, ethylene and brassinosteroids (De et al., 2005). Ethylene is an important signal for the development of roots and was reported to enhance its inhibition of Arabidopsis roots by up-regulating auxin biosynthesis (Swarup et al., 2007). Moreover, the ethylene level in the primary roots of maize was also increased after application of exogenous brassinolide (Sun et al., 2002). In carrot plant, the growth and development of roots maybe controlled by BRs, and other plant hormones, auxin, ABA, GA3, and ethylene, and so on.

Phytohormone is important for plant, but the concentration of treatment should be appropriate. In Arabidopsis, low concentration of BL can promote the growth of root, but high concentrations will decrease it (Gonzálezgarcía et al., 2011). In present study, 24-EBL at 0.5 mg/L was proved to have a positive impact on carrot petiole growth. In conclusion, this study supports the idea that BRs play an important role in regulating plant height. Carrot plants exposed to exogenous 24-EBL were relatively higher than those treated with water alone. Exogenous 24-EBL can speed up the growth of petioles in carrots.

Conceived and designed the experiments: A-SX, FQ. Performed the experiments: FQ, G-LW, FW, Z-SX. Analyzed the data: FQ, G-LW. Contributed reagents/materials/analysis tools: A-SX. Wrote the paper: FQ. Revised the paper: A-SX, G-LW. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.