Introduction
============

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 ([@B26]; [@B13]). 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 ([@B2]; [@B64]; [@B28]; [@B6]). Moreover, the roots of BR deficient mutant were found significantly shortened ([@B39]). In *Arabidopsis*, BR was found working cooperatively with auxin in promoting petiole elongation to response the shade stimulus ([@B33]). BRs were also reported to be involved in stem elongation, cell division, vascular differentiation, and leaf bending and epinasty ([@B65]; [@B14]; [@B68]; [@B29]). In field production, BRs are believed to improve crop yield and plant tolerance against environmental stress ([@B16]).

To date, more than 70 kinds of BRs have been identified ([@B36]). Among them, brassinolide (BL) is recognized as the most active form ([@B22]). 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 ([@B44]). Numerous studies have shown that regulation of genes involved in BR biosynthesis pathway can result in alteration in BR accumulation and plant growth ([@B79]; [@B81]). 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 ([@B9], [@B12]; [@B43]; [@B17]; [@B47], [@B46]; [@B45]). 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 ([@B9], [@B11]).

The homeostasis of BRs is regulated by biosynthesis genes and catabolism genes ([@B63]). In *Arabidopsis, BAS1/CYP734A1* is a major BR inactivating gene ([@B41]). In many higher plants, the *CYP734A* paralogs was also found ([@B66]). 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 ([@B72]; [@B59]; [@B78]; [@B54],[@B55]; [@B80]; [@B31]). Among these genes, *BRI1* works as the receptor for BRs, *BZR1*and *BES1* are the transcription factors that regulate the growth of plants ([@B23]; [@B27]).

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 ([@B35]; [@B77]; [@B71]). It is one of the most economically important members of *Apiaceae* plants, and the cultivated area of carrot is progressively increasing worldwide ([@B4]).

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.

Materials and Methods {#s1}
=====================

Plant Material and Exogenous 24-EBL Treatment
---------------------------------------------

'Kurodagosun' was selected as the experimental material and cultivated in a climate-control chamber at Nanjing Agricultural University (32°04′N, 118°85′E). Ambient temperature was held at 25°C for 16 h during daytime and 18°C for 8 h in the dark. The mix vermiculite and organic soil (1:1, v/v) was used to cultivated plants. The carrot samples were collected at 20 (stage 1), 40 (stage 2), 60 (stage 3), 75 (stage 4), and 90 (stage 5) days after sowing (DAS). The five stages were classified based on dates and morphological characteristics. The roots, petioles, and leaf blades were separately collected at different stages, and stored at -80°C for molecular research.

To investigate the potential effect of BR on carrot, 40 DAS carrot plants were treated with 24-EBL. The 24-EBL (≥ 85 %, Sigma--Aldrich) was dissolved in ethanol and then diluted by distilled water to make mother liquor. Four concentrations of exogenous 24-EBL (0, 0.1, 0.5, and 1 mg/L) were used, respectively. Plants treated with water alone were used as control (CK). Treatment was carried out every 2 days, five times in total. Afterward, the plants were allowed to grow for another 20 days and harvested for morphology determination.

Assay of Bioactive BR Levels
----------------------------

Samples were ground in a mortar with 10 mL of 80% methanol extraction solution containing 1 mM butylated hydroxytoluene. The mixture was incubated for 4 h at 4°C. The samples were then centrifuged for 10 min at 3500 *g*. The supernatants were filtered through a C~18~-Sep-Pak cartridge (Waters, Milford, MA, United States), and the efflux was collected and dried with N2. The mixture was dissolved in 2 mL of PBS containing 0.1 % (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5). The samples were analyzed *via* indirect enzyme-linked immunosorbent assay according to the methods described previously ([@B60]; [@B50]). Briefly: (1) The calibrating samples (epibrassinolide, CAS: 72962-43-7) or test samples (150 μL per well) were put in wells of the plate with the immobilized antibodies. Plates were placed at 37°C for 30 min. Then, removed the liquid from the wells and washed plates four times with washing buffer. (2) The Horseradish peroxidase (HRP)-conjugate (150 μL) was placed in the wells and placed at 37°C for 30 min. Then, removed the liquid from the wells and washed plates four times with washing buffer. (3) Added TMB solution (containing H~2~O~2~) to the wells and placed the plates at 37°C for 20 min. (4) Quenched the reaction by adding 2 mol/L H~2~SO~4~ (50 μL) into each well. (5) Measured optical absorbance at 450 nm. (6) Calculated the concentration according to the calibration curve. The epibrassinolide and other related brassinolide analogs were measured in this study.

Total RNA Isolation
-------------------

An RNA extraction kit (Tiangen, Beijing, China) was used to extract the total RNA of carrot roots, leaf blades, and petioles according to the manufacturer's instructions. cDNA was synthesized using a PrimerScript RT reagent kit (TaKaRa, Dalian, China). The cDNA was diluted 15-fold for quantitative reverse transcription PCR (qRT-PCR) analysis.

qRT-PCR Analysis
----------------

Genes involved in BR biosynthesis and signal transduction pathway were selected from CarrotDB^[1](#fn01){ref-type="fn"}^ ([@B76]). The primers of each gene were designed via Primer Premier 5.0 software, and were displayed in **Table [1](#T1){ref-type="table"}**. qRT-PCR was performed in a real-time PCR detection system (Bio-Rad, Hercules, CA, United States). The cycling conditions were maintained as follows: 94°C for 30 s, 40 cycles at 94°C for 10 s, 58°C for 20 s, and 61 cycles at 65°C for 10 s to create a melting curve. The experiments were performed with three independent biological replicates. Under normal growing condition, the *DcACTIN* gene was selected as the internal control to analyze gene expression and data of *DcDWF4* in carrot petioles at 90 DAS were chosen as a calibrator for gene expression analysis ([@B67]). By contrast, in the analysis of gene expression after application of 24-EBL, the *DcTUB* gene was selected as the internal control. The expression of *DcTUB* gene was more stable under abnormal growth conditions.

###### 

Oligonucleotide sequences of genes involved in the biosynthesis, catabolism, and signaling pathway.

  Gene name      Oligonucleotide sequences (5′→3′)
  -------------- -------------------------------------------
  *DcDWF4*       Forward primer: AAACGCTAAGGCTGGGCAATGT
                 Reverse primer: GCACGGCTGCAATCACTGGAA
  *DcCPD*        Forward primer: TCCCACCTACCGTAAAGCCATT
                 Reverse primer: ATCATTCTTCCTCTCCCCTCCT
  *DcDET2*       Forward primer: TCAACGCCTCTCTCCTCACTCT
                 Reverse primer: CCAGCCCTTACGGTGGTGTTT
  *DcCYP90C1*    Forward primer: GGTATGGCGAAAACAGGAGAGA
                 Reverse primer: AACACGAGATAGAGTGGCAGGG
  *DcCYP85A2*    Forward primer: AGCCACTTACATTTAATCCCTG
                 Reverse primer: TCTCCTCCAACTTCTTCCCATC
  *DcDWF5*       Forward primer: AGATGGTGGTGAAGGAGGAGAA
                 Reverse primer: CACGCAGTCATAGTGGGTTTTG
  *DcDWF1*       Forward primer: TCCGACCTTTTCTACGCTATTC
                 Reverse primer: TGTAACCCTGTGCCAGTTCTTT
  *DcBAK1*       Forward primer: TAGCACCCGAGTACCTATCCAC
                 Reverse primer: AGCAACATCACATCGTCATCAT
  *DcBRI1*       Forward primer: GAAAAGGAGGAAGAAGAAGGAA
                 Reverse primer: CAAGAAGATCTGCAAAAGTGAG
  *DcBSK1*       Forward primer: TGGCTGCTCTAGCTTAGTCTCC
                 Reverse primer: TTCCTTAGTTGCTCCAATGTGT
  *DcBZR1*       Forward primer: AGTTTCCAGCCATCGCCGTCCC
                 Reverse primer: CTGCTCCCCAGCCCAATCCTCC
  *DcBSU1*       Forward primer: TCAGCTTTTTAACTATCTTCCA
                 Reverse primer: ACCTTCTACACTATCATTTTCT
  *DcBIN2*       Forward primer: TTGTTCCCACTGTTTCTTTGATG
                 Reverse primer: TGTAGATAGCCCACTTTGTCTCC
  *DcACTIN*      Forward primer: AGAAGCACCACTGAATCCTAAGGC
                 Reverse primer: GCATACACCATCACCAGAGTCCAA
  *DcTUB*        Forward primer: CGGTATTGTGTTGGACTCTGGTGAT
                 Reverse primer: CAGCAAGGTCAAGACGGAGTATGG
  *DcCYP734A1*   Forward primer: TGCCATTCAGCCTTGGAGTA
                 Reverse primer: TGCGGATAAAGAAGCATCAACACC

Statistical Analysis
--------------------

Difference in the BRs bioactive content and the expression levels of genes in different organs during carrot development were detected by Duncan's multiple-range test at a 0.05 probability level. Student's *t*-test was used to identify the differences under different concentration treatments at the 0.05 significance.

Results
=======

Plant Growth Analysis
---------------------

The plant materials were sampled at five stages (20--, 40--, 60--, 75--, and 90 DAS) (**Figure [1](#F1){ref-type="fig"}**). The root was white at the first stage, and turned orange at the second stage. The weight of roots, petioles, and leaf blades remarkably increased between stages 2 to 3. The root continued to enlarge, and this trend was maintained at the remaining stages.

![Growth status of carrots from five different developmental stages in this research. **(A)** Stage1, 20 days after sowing, **(B)** stage2, 40 days after sowing, **(C)** stage3, 60 days after sowing, **(D)** stage4, 75 days after sowing, **(E)** stage5, 90 days after sowing. Black lines in the lower right corner of each plant represent 2 cm in that pixel.](fpls-08-01356-g001){#F1}

Changes in Bioactive BR Contents
--------------------------------

The levels of bioactive BRs in different tissues at five different stages were measured (**Figure [2](#F2){ref-type="fig"}**). BR accumulation differed in different carrot tissues and at different developmental stages.

![Bioactive BR levels in different tissues during carrot growth and development. Error bars represent standard deviation (SD). Columns with the same letter are not significantly different (*P* \< 0.05).](fpls-08-01356-g002){#F2}

In the roots, the levels of bioactive BRs were highest at first stage followed by a decrease until stage 3. The BRs levels underwent a slight increase at stage 4 and were constant until the last stage. In the petioles, the highest level of BRs was detected at 75 DAS (stage 4), whereas the lowest level was found at 60 DAS (stage 3). In the leaf blades, the level of BRs was peaked at the third stage.

Effects of 24-EBL on the Growth and Development of Carrot
---------------------------------------------------------

To determine the potential roles of BR during the development of carrot, 40 DAS (stage 2) carrot plants were treated with four different concentrations of exogenous 24-EBL (0, 0.1, 0.5, and 1 mg/L) (**Figure [3](#F3){ref-type="fig"}**). Six physiological indexes including total plant height, root length, root diameter, root weight, aboveground weight, and number of petioles were selected for comparison among the treatments. Under different concentrations of 24-EBL treatments, the root length, root diameter, and root weight did not show obvious difference compared with control. However, 24-EBL obviously increased the total plant height of carrot (**Figures [4A,B](#F4){ref-type="fig"}**). When carrot plants were treated with 0.5 mg/L 24-EBL, the aboveground weight of carrot was significantly increased compared to the control (**Figure [4B](#F4){ref-type="fig"}**). Similarly, carrot plants exposed to 0.5 mg/L 24-EBL displayed increased number of petioles when compared with control plants (**Figure [4C](#F4){ref-type="fig"}**).

![Growth status of carrots treated with exogenous BL. 40 days after sowing plants were treated with different concentrations of BL (0, 0.1, 0.5, and 1 mg/L). White lines in the lower right corner of each plant represent 5 cm.](fpls-08-01356-g003){#F3}

![Exogenous BL induces petiole growth in carrot. 40 days after sowing plants were treated with different concentrations of BL (0, 0.1, 0.5 and 1 mg/L). **(A)** Root length, overall length, and root width; **(B)** root weight and aerial part (petioles and leaf blades) weight; **(C)** the number of petioles. Student's *t*-test was used to identify the differences under different treatments (*P* \< 0.05; ^∗^, control versus treatment). Error bars represent standard deviation (SD).](fpls-08-01356-g004){#F4}

Expression Profiles of Genes Involved in BR Biosynthesis and Catabolism
-----------------------------------------------------------------------

*DcDWF7, DcDWF1, DcDET2, DcDWF4, DcCPD, DcCYP85A2, DcCYP90C1*, and *DcCYP734A1* were recognized as genes involved in BR biosynthesis and catabolism and their expression levels in carrot roots, petioles and leaf blades at five developmental stages were measured by qRT-PCR (**Figure [5](#F5){ref-type="fig"}**) ([@B44]; [@B8]).

![Expression profiles of genes involved in the BR biosynthesis and catabolism pathway at different stages and in different organs. Error bars represent standard deviation (SD). Columns with the same letter are not significantly different (*P* \< 0.05).](fpls-08-01356-g005){#F5}

### In the Roots

*DcDWF4*, a key gene in BR biosynthesis, showed the highest expression level at stage 1 and the lowest level at stage 4. *DcCPD* and *DcCYP90C1* peaked at stage 1 followed by a decrease. Afterward, the highest level of *DcDET2* appeared at stage 5. *DcDWF1* showed higher expression as compared with other genes. *DcCYP734A1* is a catabolism gene of BRs and showed significantly higher expression level at stage 1 compared with other stages.

### In the Petioles

The minimum expression level of the five genes (i.e., *DcDWF4, DcCPD, DcDET2, DcCYP90C1*, and *DcCYP85A2*) was observed at stage 3 or 4. The five genes exhibited similar expression trends. *DcDWF7* and *DcDWF1* were lowly expressed at the last stage. Transcription level of *DcDWF1* was relatively higher than that of others. The expression level of catabolism gene *DcCYP734A1* peaked at stage 1.

### In the Leaf Blades

*DcDWF7, DcDET2*, and *DcCYP85A2* were highly expressed at third stage. *DcDWF1, DcDWF4*, and *DcCYP90C1* showed a similar expression pattern which peaked at first stage and gradually decreased afterwards. In the leaf blades, *DcCYP734A1* gene showed the highest expression level at stage 3.

Expression Profiles of Genes Involved in the BR Signaling Pathway
-----------------------------------------------------------------

In this study, transcript levels of *DcBAK1, DcBRI1, DcBSK1, DcBZR1, DcBSU1*, and *DcBIN2*, which have been reported to be involved in the BR signaling pathway, were analyzed using qRT-PCR ([@B3]; [@B73]; [@B24]). The expression profiles are shown in **Figure [6](#F6){ref-type="fig"}**.

![Expression profiles of genes involved in the BR signaling pathway at different stages and in different organs. Error bars represent standard deviation (SD). Columns with the same letter are not significantly different (*P* \< 0.05).](fpls-08-01356-g006){#F6}

### In the Roots

*DcBRI1* and *DcBAK1* showed higher expression levels at the first two stages and relatively lower expression levels at later stages. The expression levels of *DcBSK1, DcBIN2, DcBSU1*, and *DcBZR1* increased at second stage and declined at the third stage, and then increased at stage 4 and decreased at stage 5.

### In the Petioles

*DcBRI1* and *DcBSU1* were highly expressed at the fourth stage, whereas the lowest expression level of *DcBZR1* was detected at this stage. *DcBAK1, DcBSK1, DcBZR1* showed highest expression level at the last stage. The highest transcription of *DcBIN2* was observed at the first stage, followed by a continuous decrease at the remaining stages.

### In the Leaf Blades

*DcBRI1, DcBSK1, DcBSU1, DcBZR1*, and *DcBIN2* showed higher expression levels at stage 3. *DcBAK1* showed relatively stable expression level at the first two stages, followed by a obvious decrease at the later stages.

Effects of 24-EBL Application on the Expression Level of BR Biosynthetic and Catabolism Genes
---------------------------------------------------------------------------------------------

To study the effect of 24-EBL on BR biosynthesis, we investigated the expression level of related genes. The expression levels of these genes under different BL concentrations were measured by quantitative reverse transcription PCR (qRT-PCR). These genes were strongly regulated by exogenous BL treatment (**Figure [7](#F7){ref-type="fig"}**).

![Expression profiles of genes involved in the BR biosynthesis and catabolism pathway under different BL concentrations (0, 0.1, 0.5, and 1 mg/L) and in different organs. Error bars represent standard deviation (SD). Student's *t*-test was used to identify the differences under different concentration treatments (*P* \< 0.05; ^∗^, control versus treatment).](fpls-08-01356-g007){#F7}

### In the Roots

All the genes examined were significantly up-regulated at the 0.5 mg/L. In the presence of 1 mg/L 24-EBL, *DcDWF1, DcCPD*, and *DcCP90C1* showed lower expression levels as compared with control plants. When compared with control group, *DcDWF4* expression level was significantly reduced in the roots treated with 0.1 mg/L BL, whereas transcription of *DcCYP85A2* and *DcCYP90C1* was increased.

### In the Petioles

The expression levels of *DcDET2, DcDWF4, DcCPD, DcCYP85A2*, and *DcCYP90C1* were significantly down-regulated after application of exogenous BL. In the expression profile of *DcDWF7*, expression level did not show significant change with 0.1 and 1 mg/L 24-EBL, but the expression level was significantly up-regulated with 0.5 mg/L. The catabolism gene *DcCYP734A1* was significantly up-regulated under the treatment of 0.5 mg/L 24-EBL.

### In the Leaf Blades

The expression levels of *DcCPD, DcCYP90C1*, and *DcCYP85A2* was down-regulated after application of 24-EBL. Meanwhile, the expression levels of *DcDWF7* and *DcDET2* did not show significant change. However, *DcDWF1* showed up-regulation trend with 0.1 and 0.5 mg/L and *DcDWF4* was up-regulated by 0.5 mg/L 24-EBL. The expression level of the catabolism gene *DcCYP734A1* was significantly up-regulated under the treatment of 0.5 mg/L 24-EBL.

Effects of 24-EBL Treatment on the Expression Profiles of BR Response Genes
---------------------------------------------------------------------------

To further investigate the effect of 24-EBL application on the expression levels of BR response genes, six genes were selected and their expression profiles were evaluated by qRT-PCR. Most genes were strongly regulated after the treatment (**Figure [8](#F8){ref-type="fig"}**).

![Expression profiles of genes involved in the BR signaling pathway under different BL concentrations (0, 0.1, 0.5, and 1 mg/L) and in different organs. Error bars represent standard deviation (SD). Student's *t*-test was used to identify the differences under different concentration treatments (*P* \< 0.05; ^∗^, control versus treatment).](fpls-08-01356-g008){#F8}

### In the Roots

The expression levels of all genes was increased in the presence of 0.5 mg/L BL. *DcBRI1*, and *DcBSK1* expression levels was up-regulated in the plants treated with 0.1 and 0.5 mg/L BL. However, their expression levels in the presence of 1.0 mg/L showed no significant difference as compared with control group. *DcBAK1, DcBIN2, DcBSU1*, and *DcBZR1* were up-regulated at all the three concentrations 24-EBL levels.

### In the Petioles

Most genes showed significant up-regulation at the concentration of 0.5 mg/L, whereas *DcBSK1* expression level showed no significant change. The expression levels of *DcBSK1* and *DcBSU1* was down-regulated when treated with 0.1 and 1 mg/L. Transcription of *DcBZR1* was significantly increased in the presence of 1 mg/L BL.

### In the Leaf Blades

Most genes (except *DcBAK1* and *DcBSU1*) were up-regulated in the presence of 0.5 mg/L BL. When treated with 1 mg/L BL, the expression level of most genes significantly decreased, whereas *DcBZR1* showed no obvious change. *DcBAK1* and *DcBIN2* expression levels in plants treated with 0.1 and 0.5 mg/L BL did not differ significantly from control.

Discussion
==========

Phytohormones are very important factors affecting almost all the processes of plant growth ([@B57]). 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 ([@B51]; [@B14]; [@B34]; [@B2]; [@B53]). Functions of BRs have been documented in many plants, such as, tomato, cucumber, rice, and maize ([@B37]; [@B21]; [@B32]). 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 ([@B57]). In addition, the roles of a specific hormone also depend on the presence of other phytohormone and plant growth regulators ([@B42]). 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 ([@B56]; [@B37]).

In carrots, the content of bioactive BR levels at different stages and in different organs keeps changing (**Figure [2](#F2){ref-type="fig"}**). 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 ([@B50]). 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) ([@B5]; [@B71], [@B70]; [@B75]).

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 ([@B38]). In rice, *Dwarf1* gene was found to encode GTP-binding protein. The phenotype of its recessive mutant is dwarf ([@B34]; [@B52]; [@B1]). According to previous reports, the dwarf phenotype of *dwarf* mutants was associated with brassinosteroids and gibberellins ([@B30]). 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 ([@B30]). By contrast, exogenous BL stimulated the unrolling of leaf blades in wheat ([@B69]). In *Arabidopsis*, BR-deficient mutants appeared to have stunted shoot and small leaves ([@B40]). 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 ([@B20]). 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* ([@B40]). Similar results were also observed in rice ([@B30]). Plant height is an important index for plant growth. BR was reported to be a crucial factor for plant height ([@B30]). 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 ([@B48]). 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 ([@B10]; [@B7]; [@B20]; [@B49]). 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 ([@B25]; [@B57]). 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 ([@B19]; [@B64]). 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 ([@B62]). In tomato, the long-distance transport of BRs is also lacking ([@B37]). Moreover, BRs were thought to regulate root meristem size maintenance depending on their action site ([@B74]). In *Arabidopsis*, the meristem size of root was reported to be controlled by BR signaling in the root epidermis ([@B25]). 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 ([@B42]). In rice, the signaling pathways of auxin, ABA and GA3 controlling cell elongation are affected by other pathway involving brassinosteroids ([@B18]). In *Arabidopsis*, the growth of hypocotyls was reported to be controlled by auxin, ethylene and brassinosteroids ([@B15]). 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 ([@B61]). Moreover, the ethylene level in the primary roots of maize was also increased after application of exogenous brassinolide ([@B58]). 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 ([@B25]). 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.

Author Contributions
====================

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.

Conflict of Interest Statement
==============================

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.

The research was supported by the New Century Excellent Talents in University (NCET-11-0670); Jiangsu Natural Science Foundation (BK20130027); Priority Academic Program Development of Jiangsu Higher Education Institutions.

<http://apiaceae.njau.edu.cn/carrotdb/index.php>

24-EBL

:   24-epibrassinolide

*ACTIN*

:   Actin 1 gene

BAK1

:   BRI1-associated receptor kinase1

BES1

:   BRI1-EMS-suppressor1

BIN2

:   bridging integrator 2

BRI1

:   Brassinosteroid insensitive 1

BRs

:   brassinosteroids

BSK1

:   BR-signaling kinase1

BSU1

:   BRI1 suppressor 1

BZR1

:   brassinazole resistant1

*CPD*

:   *CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM*

DAS

:   days after sowing

*DET2*

:   DE ETIOLATED2

*DWF1*

:   *DWARF1*

DWF4

:   *DWARF4*

*DWF7*

:   *DWARF7*

*TUB*

:   Tubulin beta-7 gene

qRT-PCR

:   quantitative reverse transcription PCR

[^1]: Edited by: *Elena Prats, Consejo Superior de Investigaciones Científicas (CSIC), Spain*

[^2]: Reviewed by: *Andrzej Bajguz, University of Białystok, Poland; Hao Peng, Washington State University, United States*

[^3]: This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science
