Plant hormones are essential elements that control a great number of cellular processes that occur during plant growth. Abscisic acid (ABA) is a plant hormone known for its roles in regulating growth/development and response to biotic/abiotic stresses of the plant. It modulates plant growth and development in many aspects, such as seed dormancy, stomata movement, and xylem fiber differentiation [1]. Furthermore, ABA can inhibit stem elongation and shoot growth, as well as reduce shoot weight, which results in the repression of the overall growth of the plant. It has been postulated that ABA inhibits shoot growth by restricting the synthesis of gibberellin [2].
The carrot (Daucus carota L.), a plant of the Apiaceae family, is one of the most important vegetable crops, and it is also highly appreciated worldwide for its abundance of nutrients, including pro-vitamin A and carotenoid [3,4]. Carrots are cultivated all over the world. The total cultivated area of the carrot crop in the world is more than 1 million hectares per year, and China is the No. 1 carrot crop producer globally. Carrot yield and quality are significantly affected by carrot growth and development. The formation and enlargement of the fleshy roots is a complex process involving structural changes, material accumulation, and gene regulation, which is directly or indirectly regulated by plant hormones [5].
Alongside the cellulose in the root of the carrot, lignin is another complex organic polymer within the plant. It provides the required mechanical strength for the cells and tissues of vascular plants that participate in transporting water and nutrients between different parts [6]. Plants contain an outsized quantity of lignin within the vascular tissue of their roots. However, an excessive amount of lignin can negatively influence the growth and development of the carrot taproot, thus decreasing the quality. Therefore, the lignin content of the carrot root is a crucial indicator of the quality of this root vegetable crop [7,8].
However, the relationships among ABA biosynthesis, accumulation, and the signal transduction occurring during growth and lignification of carrots have not been explicitly discussed. The effects of ABA on cambium differentiation and lignification in carrot roots were seldom studied. Therefore, in the present work we examined the potential role played by exogenous ABA in the physiological growth, root anatomical structure, and accumulation of lignification in carrots.
The growth, anatomical structure, and lignification of carrot roots (Kurodagosun, Musashino Seed Co., Ltd, Tokyo, Japan) were studied in a control group alongside plant groups treated with three concentrations (10, 50, and 100 μM) of ABA (Beijing Solarbio Science & Technology Co., Ltd, Beijing, China). As shown in Fig. 1A,B, ABA application resulted in a significant decrease in both total plant length and shoot length, whereas there were no statistically significant changes observed in the diameter of carrot roots. Moreover, it was observed that the shoot weight of carrot plants was significantly reduced compared to the control group. However, there were no significant changes in the root weight of carrot plants across all four groups. The petiole number of carrot seedlings in the control group showed no significant changes compared to plants that were treated with 10 μM ABA, whereas plants treated with 100 μM ABA showed a significant reduction in the number of petioles compared to the control group. These results indicated that exogenous ABA affected the growth of carrot shoot, which was in agreement with previous findings. It has been confirmed that increased ABA in leaves and xylem sap could inhibit leaf growth [2].
Effects of exogenous ABA on carrot root growth (A) Plant growth of carrots treated with different concentrations of ABA. White lines in the lower right corner of each plant represent 5 cm in that pixel. (B) Morphological characteristics of carrots treated with different concentrations of ABA. (C) Anatomical structure of carrot roots treated with ABA. The central part of root cross section is showed. Scale bars represent 100 μm in length. (D) Influence of exogenous ABA on lignin content in carrot roots. Data were presented as the mean ± standard deviation (SD). Different letters (a, b, and c) indicate significant differences at P < 0.05.
Expression profiles of genes involved in lignin or ABA biosynthesis in carrot roots treated with different concentrations of ABA (A) Expression profiles of genes involved in lignin biosynthesis and polymerization in carrot roots treated with ABA. (B) Expression profiles of genes involved in ABA biosynthesis in carrot roots treated with different concentrations of ABA. Data were presented as the mean ± standard deviation (SD). Different letters (a, b, c, and d) indicate significant differences at P < 0.05.
The transverse sections of carrot roots treated with different concentrations of ABA and those in the control group were taken and stained with safranin O and fast green to highlight the underlying anatomical structure of the carrot roots (Fig. 1C). It was observed that there were thinner cell walls around the parenchyma in the secondary xylem under ABA treatment. Furthermore, fluorescence micrographs of the transverse sections of the carrot roots treated with ABA were analyzed to explore the effect of exogenous ABA treatment on carrot root lignification (Supplementary Fig. S1). Decreased accumulation of lignin could be found in ABA-treated carrot roots. Poor lignification was exhibited under UV light, and comparatively lower lignification was found in ABA-treated groups compared to the control group. The lignin content in roots of carrot treated with ABA was analyzed to examine ABA’s role in the lignification. Exogenous ABA significantly decreased the content of lignin in carrot roots. The root lignin content was 8.95 mg/g in the control group. Whereas the root lignin content was 6.42, 5.43, and 6.15 mg/g in the carrots treated with 10, 50, and 100 μM ABA, respectively (Fig. 1D).
The above results highlighted that exogenous ABA-treated groups exhibited reduced plant and root growth, as well as a decrease in secondary xylem development and lignification, which indicated the role of ABA in lignin biosynthesis and the polymerization of carrot roots. Therefore, we searched the nucleotide sequences of genes encoding key enzymes in lignin synthesis and polymerization from a carrot genomic and transcriptomic database Carrot DB (http://apiaceae.njau.edu.cn/carrotdb) to investigate whether these morphological and anatomical changes in carrot roots are due to the altered gene expression patterns. The key enzymes in lignin biosynthesis and polymerization that we determined by RT-qPCR were as follows: phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), p-coumaroyl shikimate/quinate 3′-hydroxylase (C3′H), carbon dioxide (COMT), hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), peroxidase (PER), and laccase (LAC). Total RNA of the carrot samples was extracted and reverse transcribed into cDNA, both of which were identified by Nano-Drop for determining their qualities and concentrations. Two carrot reference genes, DcActin and DcTUB, were selected to normalize the gene expression levels [9]. The results demonstrated that exogenous ABA leads to a down-regulated expression pattern in most of the genes involved in lignin biosynthesis (Fig. 2A), especially the DcPAL gene. Similarly, the DcC4H gene showed a significantly lower expression in carrots treated with 10, 50, and 100 μM ABA as compared to the control group. In the case of the Dc4CL gene, almost trace expression levels were observed under 10 and 50 μM ABA treatment as compared to the control group. However, all these genes showed functional expression in the control plants.
Moreover, the DcF5H gene showed no significant changes in the expression level in the plants treated with 10 or 50 μM ABA as compared to the control group. Nevertheless, its expression level was highly up-regulated in the seedlings treated with 100 μM ABA. The DcCAD gene was observed to be highly up-regulated, and the expression level was significantly increased with a higher dosage of ABA. The DcHCT gene exhibited nil expression in the plants treated with 10 and 50 μM ABA but was highly expressed under the treatment with 100 μM ABA compared to the control group. The expressions of DcCOMT and DcCCoAOMT exhibited significant down-regulation in ABA-treated groups. The expression level of all these genes was found to be subsequently reduced with increased doses of ABA as compared to the control group. The DcC3′H gene was highly down-regulated under 10 μM ABA treatment as compared to the control group. However, plants treated with 50 and 100 μM ABA, respectively, showed no significant changes in gene expression as compared to the control group. The DcCCR gene was found to be down-regulated under all ABA treatments in comparison to the control group.
The expression levels of four genes involved in lignin polymerization, i.e. DcPER1, DcPER2, DcLAC1, and DcLAC2, were also detected. DcPER1 showed a significant down-regulation under the doses of 50 and 100 μM ABA, but there was no significant difference between the treatment with 10 μM ABA and the control. DcPER2 showed a down-regulation under 10 μM ABA, but was up-regulated by the treatment with 100 μM ABA. DcLAC1 and DcLAC2 were significantly down-regulated in all the groups.
The key genes involved in ABA biosynthesis, i.e. DcPSY1, DcPDS, DcZDS2, DcLCYB1, DcCHXB1, DcZEP, DcNXS, DcNCED3, and DcABA2, were chosen for expression pattern analysis in the carrot roots, leaf blades, and petioles of control and ABA-treated plants (Fig. 2B). The expression levels of all the genes, DcPSY1, DcPDS, DcZDS2, DcLCYB1, DcCHXB1, DcZEP, DcNXS, DcNCED3, and DcABA2, were found to be up-regulated in the carrot roots, leaf blades, and petioles in ABA-treated plants as compared to the control group.
In summary, the present study was an effort to demonstrate the roles of exogenous ABA on carrot growth traits, as well as the lignification of the carrot roots. ABA suppressed the lignification in the carrot roots due to the down-regulation of responsible lignin biosynthetic and polymerization genes. The tissue-specific expression of ABA biosynthesis genes and their enhanced expression in leaf blade tissues are in line with the previous studies. Therefore, ABA not only repressed the growth and altered root anatomical structure of carrots but also reduced the secondary xylem lignification by influencing the regulation of genes involved in lignin biosynthesis. These findings provide a new insight into further studies for a better understanding of the molecular mechanisms of lignin biosynthesis regulation in carrots.
Funding
This work was supported by the grants from the National Natural Science Foundation of China (No. 31872098) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
References
Author notes
Ahmed Khadr and Yahui Wang contributed equally to this work.
