Gibberellins (GAs) are considered potentially important regulators of cell elongation and expansion in plants. Carrot undergoes significant alteration in organ size during its growth and development. However, the molecular mechanisms underlying gibberellin accumulation and perception during carrot growth and development remain unclear. In this study, five stages of carrot growth and development were investigated using morphological and anatomical structural techniques. Gibberellin levels in leaf, petiole, and taproot tissues were also investigated for all five stages. Gibberellin levels in the roots initially increased and then decreased, but these levels were lower than those in the petioles and leaves. Genes involved in gibberellin biosynthesis and signaling were identified from the carrotDB, and their expression was analyzed. All of the genes were evidently responsive to carrot growth and development, and some of them showed tissue-specific expression. The results suggested that gibberellin level may play a vital role in carrot elongation and expansion. The relative transcription levels of gibberellin pathway-related genes may be the main cause of the different bioactive GAs levels, thus exerting influences on gibberellin perception and signals. Carrot growth and development may be regulated by modification of the genes involved in gibberellin biosynthesis, catabolism, and perception.
Plant growth and development is a complex process that involves differentiation and morphogenesis. Hormonal regulation is recognized as a key process in plant growth and development1. For example, gibberellins (GAs) are phytohormones that play essential roles in plant growth and developmental stages, including seed germination, flowering, sex expression, and leaf and fruit senescence2,3. It has been reported that GA-mediated plant growth is mainly achieved by promoting cell elongation in plants4. Further studies indicate that GAs also regulate cell production to control plant growth5. Inadvertently, GA was first discovered in the fungus Gibberella fujikuroi 6. To date, more than 100 GAs have been identified in plants, fungi, and bacteria; however, only a few GAs exhibit biological activities. GA1, GA3, GA4, and GA7 are the major bioactive GAs; other GAs are non-bioactive and act as precursors of the bioactive forms7. To understand the roles of GAs in plant growth and development, researchers should investigate the regulation and response of GAs in plants.
In higher plants, GAs originate from geranylgeranyl diphosphate (GGPP), which is synthesized from isopentenyl pyrophosphate (IPP)8. GGPP is then converted into ent-kaurene by ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Ent-kaurene is oxidized to GA12-aldehyde, a precursor of all GAs. Finally, GA12-aldehyde is then transformed into various GAs in a process catalyzed by GA20-oxidase (GA20ox), GA3-oxidase (GA3ox), GA2-oxidase (GA2ox), and other enzymes9,10 (Figure 1). Biochemical, molecular, and genetic studies have shown that genes that encode the enzymes in this pathway are essential for GA accumulation and plant growth11,
Hormone-mediated control of plant growth and development involves both synthesis and response17. DELLA proteins are major inhibitors of plant growth and development18. A bioactive GA binds to a GA receptor, namely, GIBBERELLIN INSENSITIVE DWARF1 (GID1), and forms a GA-GID1 complex; as a result, the degradation of DELLAs is triggered19,20. This mechanism is also called de-repression of GA. Further studies on Arabidopsis and rice have found that a specific ubiquitin E3 ligase complex (SCFSLY1/GID2) is required for this process21. In recent years, positive and negative factors of GA signaling were identified in higher plants22. SLEEPY1 (SLY1), GAMYB, and PICKLE act as positive regulators of gibberellin signaling23,24. Negative regulators of gibberellin signaling include SHORT INTERNODE (SHI), SPINDLY (SPY), and other proteins25,26. Chitin-inducible gibberellin-responsive protein (CIGR) may be involved in GA-mediated phosphorylation/dephosphorylation of DELLAs27.
Carrot (Daucus carota L.), an Apiaceae plant, is commonly consumed worldwide for its nutritional value28. Carrot has also been the focus of many studies aimed at investigating the molecular biology of plants29,
Materials and methods
Plant material and growth conditions
The seeds of D. carota L. cv. ‘Kurodagosun’ were cultivated in an artificial chamber at the Nanjing Agricultural University (32°02′N, 118°50′E). The plants were grown at 25°C for 16 h during the daytime followed by 18°C for 8 h in the dark. Samples were collected at 25, 42, 60, 75, and 90 days after sowing (DAS). Morphological characteristics and age were considered to verify the developmental stages. To ensure the accuracy of biochemical and molecular research, whole carrot taproot was collected at each developmental stage. For upper ground tissues, petioles and leaves from whole carrot plants were separately harvested at each developmental stage. The samples of whole taproot, petioles, and leaves were ground in a mortar. The samples were then randomly divided into two groups for GA determination and RNA isolation. Three biological replicates were performed at each collection time point.
Anatomical structure analysis
To examine the growth status of each plant, we investigated the changes in cell structure during carrot growth and development. Fresh samples were cut into small pieces and immediately stored in phosphate buffer (pH 7.2) with 2.5% glutaraldehyde. The slices were dehydrated with ethanol and then treated with epoxy propane. Subsequently, the samples were soaked and embedded in Spurr resin37. A Leica ultramicrotome (Germany) was used to cut the samples into thin sections (∼1 μm), which were stained with 0.5 % methyl violet for 10 min. Then, the slices were observed and then photographed under a Leica DMLS microscope (Germany).
Assay of bioactive GA levels
Samples were ground in a mortar with 10 mL of 80% methanol extraction solution containing 1 mM butylated hydroxytoluene. The extract was incubated at 4°C for 4 h and centrifuged at 3500g for 10 min. Supernatants were then filtered through Chromoseq C18 columns. Efflux was collected and dried with N2. Residues were dissolved in a phosphate-buffered saline (PBS) solution containing 0.1 % (v/v) Tween 20 and 0.1 % (w/v) gelatin (pH 7.5). The endogenous levels of bioactive GAs were then determined using an indirect enzyme-linked immunosorbent assay (ELISA) as previously described38,
Total RNA extraction and cDNA synthesis
Total RNA was isolated from carrot roots, petioles, and leaves at different stages using an RNAsimple Total RNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. RNA quality and concentration was then assessed by gel electrophoresis and the use of a One-Drop spectrophotometer. To eliminate contaminating genomic DNA, we treated total RNA with gDNA Eraser for 2 min at 42°C (TaKaRa, Dalian, China).cDNA was synthesized using PrimeScript RT reagent kit (TaKaRa, Dalian, China) according to the manufacturer’s protocols. The cDNA was then diluted 10 times for quantitative real-time PCR (qRT-PCR) analysis.
Gene expression analysis by quantitative real-time PCR
To identify the genes involved in GA biosynthesis and response in carrot, GA-related genes of Arabidopsis and other plant species were aligned with the sequences in carrotDB, a genomic and transcriptomic database for carrot, which was built by our group (Lab of Apiaceae Plant Genetics and Germplasm Enhancement, http://apiaceae.njau.edu.cn/carrotdb/index.php)29 (Figure S1–S29). Primers used for qRT-PCR were designed with Primer Premier 6 software (Tables 1 and 2). qRT-PCR was performed using SYBR Premix Ex Taq (TaKaRa, Dalian, China). Each reaction contained 2 μL of diluted cDNA strand, 10 μL of SYBR Premix Ex Taq, 7.4 μL of deionized water, and 0.4 μL of each primer, accumulating a final volume of 20 μL. PCR was strictly performed according to the following standards: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The experiments were repeated with three independent samples, and the results were normalized against the carrot reference gene, namely, DcActin31. Data from DcGA2ox2 in carrot root at 22 DAS were selected as calibrators for gene expression analysis.
Differences in the GA levels during carrot development were detected using Duncan’s multiple-range test at a 0.05 probability level.
Plant growth analysis
Over the course of growth and development, carrot tissues were harvested at 25, 42, 60, 75, and 90 DAS, respectively (Figure 2). The developmental stages were identified by age and growth indices. The root was white, and the fresh weight of the root was less than that of the shoot at 25 DAS. Up to 42 DAS, the root surface appeared orange, and the root, together with the petioles, was evidently elongated. Root weight and diameter significantly increased between 42 and 60 DAS. Subsequently, the root continued to enlarge, and the root became heavier than the shoot. However, root length presented no evident change (Figure 2).
Structural changes in the roots, petioles, and leaves
In the roots
At 25 DAS, the root was white, and the vascular cambium (VC) located between primary phloem (PP) and protoxylem (Px) did not show evident thickness (Figure 3A). At 42 DAS, the root surface appeared orange, and VC differentiated outward and inward, forming the secondary phloem (SP) and the secondary xylem (SX), respectively (Figure 3B). Subsequently, the root continued to enlarge (Figure 3C, F, and J).
In the petioles
The collenchyma (Co), which provides structural support for carrot growth, was very conspicuous under the microscope (Figure 4). As the plant grew, regions of Co, phloem (P), and xylem(X) were evidently enlarged, suggesting that constant thickness developed in the petioles.
In the leaves
At 25 DAS, the numbers of palisade and spongy cells were limited, and the cell arrangement was disordered (Figure 5A). Subsequently, the leaves expanded, and the cells in the palisade tissue (Pt) and spongy tissue (St) were compactly arranged (Figure 5B–E).
Changes in bioactive GAs
The levels of bioactive GAs (GA1, GA3, GA4, and GA7) were analyzed in the roots, petioles, and leaves during carrot growth and development (Figure 6). For each plant, the bioactive GA contents in the leaves and petioles were higher than those in the roots. During carrot growth, the highest GA levels in the roots were observed at 42 DAS; this level subsequently decreased. However, GA levels in the petioles slightly changed, and the GA levels in the leaves relatively fluctuated.
Expression profiles of genes in the GA biosynthetic pathway during carrot growth and development
DcKS, DcKO, DcKAO1, DcKAO2, DcGA20ox1, DcGA20ox2, DcGA20ox3, DcGA3ox1, DcGA3ox2, DcGA2ox1, DcGA2ox2, DcGA2ox3, DcGA2ox4, and DcGA2ox5 were identified from our database. These genes were then selected for qRT-PCR to investigate their expression levels. Biosynthetic pathway-related genes were evidently regulated by carrot growth and development (Figure 7).
In the roots, transcript levels of DcKS, DcKO, DcKAO1, DcGA3ox1, and DcGA2ox5 exhibited a similar pattern, in which an initial increase and a subsequent decrease were observed. In contrast, the mRNA level of DcGA20ox3 initially decreased and then increased. The mRNA levels of DcKAO2, DcGA20ox1, DcGA2ox2, and DcGA2ox3 initially decreased then increased and finally decreased again. Conversely, the mRNA level of DcGA2ox1 showed completely opposite results. However, DcGA20ox2, DcGA3ox2, and DcGA2ox4 constantly decreased over the course of the experiment. In the petioles and leaves, biosynthetic pathway-related genes were also significantly regulated by growth and development.
For the same plant, the expression levels of these genes differed in different tissues. For example, the mRNA levels of DcKAO2 and DcGA3ox1 in the roots were lower than those in the petioles or leaves, whereas DcKO showed the highest level in the roots during carrot growth and development (Figure 7).
Expression profiles of GA-responsive genes during carrot growth and development
GA perception and subsequent signal transduction are essential for GA functions during plant growth. Thus, GA receptors should be identified to further increase our understanding of the GA signaling pathway. The genes involved in GA response, namely, GID1a, GID1b, GID1c, DcDELLA1, DcDELLA2, DcDELLA3, DcSLY1, DcCIGR, DcPICKLE1, DcPICKLE2, DcSPY, DcGAMYB, DcSHI1, DcSHI2, and DcSHI3 showed marked changes in mRNA levels during plant growth (Figure 8). In the roots, the transcript levels of DcGID1b and DcSLY1 were higher at 90 DAS and lower at 25 and 42 DAS. DcCIGR, DcSHI2, and DcSHI3 showed high expression at 25 and 42 DAS and consistently low expression at the last three time points. DcGID1a, DcGID1c, DcCIGR, DcPICKLE1, DcPICKLE2, DcSPY, DcGAMYB, and DcSHI1 were highly expressed at 60 DAS, whereas transcription of DcDELLA1 was highest at 75 DAS. In the petioles, transcription of DcDELLA3 was highest at 40 DAS. DcGID1b, DcGID1c, DcSLY1, DcCIGR, DcPICKLE2, DcSPY, and DcSHI1 showed high expression at 90 DAS. In the leaves, DcGID1c, DcDELLA1, DcDELLA2, DcCIGR, DcCIGR, DcPICKLE2, and DcSHI2 exhibited the highest mRNA abundance at 25DAS, whereas DcGID1a and DcGID1b showed high expression at 90 DAS.
For the same plant, some genes, including DcDELLA1, DcDELLA2, DcDELLA3, DcCIGR, DcPICKLE1, DcSPY, DcSHI1, and DcSHI3, showed the lowest expression in the roots across all growth stages. However, expression patterns of other genes in different tissues may change during plant growth. DcGID1a, DcGID1b, DcDELLA1, DcDELLA2, DcDELLA3, DcSLY1, and DcCIGR were expressed at higher levels than other genes (Figure 8).
Hormonal regulation is essential for plant growth and development41,42. Several classes of hormones have been identified, which are ascribed to growth regulation43. Among these hormones, GAs usually promote cell elongation44. Carrot, a root vegetable, is commonly consumed worldwide for its nutritional value. Carrot undergoes significant alterations in its tissues during plant growth. Our previous work suggested that some GA-related genes are differentially expressed at different carrot root developmental stages, indicating that GAs may play important roles in carrot root development29,45. Thus, GA accumulation and its potential role in carrot should be further investigated.
Carrot growth and structural development
In this study, marked elongation and differentiation was observed at 42 DAS, suggesting that this time point may be crucial for root development. Root enlargement can be attributed to the continuous differentiation of VC. A 42-day-old carrot may also be active in pigment accumulation because the root surface first appeared orange during this time46,47. The petiole, as a transport organ, was elongated and thickened during plant growth (Figure 4). The leaves also evidently expanded (Figure 5). Therefore, we aimed to investigate whether GAs are implicated in these processes and to determine the response of carrots to GAs.
GA content and functions
Our results found that the levels of bioactive GAs were higher in the leaves and petioles than in other parts (Figure 6). These findings indicated that GA biosynthesis and catabolism might be tissue-specific48. Previous studies have suggested that GAs are the major promoters of cell elongation49,
GA biosynthesis and catabolism
GAs are present in all tissues and are essential for carrot growth. However, our results found that GA levels may differ in various carrot tissues. GA levels were also significantly altered during carrot growth. Thus, GA production and regulation should be understood. In this study, the expression levels of 14 genes involved in GA biosynthesis were studied during carrot growth and development. All of these genes responded to the growth stages, thereby eliciting evident influence on the GA levels. However, the expression patterns were incompletely consistent with the GA levels possibly because of the feedback mechanisms of GAs53,
GA response and regulators
GA perception and signaling transduction are also important for GA-mediated plant growth and development. A GA-GID1-DELLA signaling module has been extensively described21,58. A total of 15 receptors or acting components in this module were investigated using qRT-PCR during carrot growth. These genes were evidently regulated by carrot growth and development, and this result indicated that this module is also present in carrot.
The transcripts of DcDELLA1, DcDELLA2, DcDELLA3, DcCIGR, DcPICKLE1, DcSPY, DcSHI1, and DcSHI3 were higher in the petioles and leaves than in the roots. This observation was consistent with the GA levels among different tissues, which indirectly suggested that bioactive GAs are produced at their site of action59,60.
Interestingly, genes encoding DELLAs in the current study were expressed at high levels. Two main factors support this finding. First, high DcDELLA transcript levels suggested that DELLAs may be the main restraints in plant growth61,62. Second, DELLA homeostasis could balance or relieve the cues induced by GAs or other hormones63, thereby stabilizing plant growth.
The morphological and anatomical structure of roots, petioles, and leaves were significantly altered during carrot plant growth. GAs may play important roles in the elongation and expansion of carrot tissues. The relative transcript levels of pathway-related genes may be the main cause for the different levels of bioactive GAs, therefore exerting influences on gibberellin perception and signals. Carrot growth and development may be regulated by modification of the genes involved in gibberellin biosynthesis, catabolism, and perception.
The research was supported by the following: New Century Excellent Talents in University (NCET-11-0670); Jiangsu Natural Science Foundation (BK20130027); the Open Project of State Key Laboratory of Crop Genetics and Germplasm Enhancement (ZW2014007); and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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