Next Article in Journal
Evaluation of Strawberry Colletotrichum spp. Genetic Diversity in Lithuania
Previous Article in Journal
Variable Rate Seeding and Accuracy of Within-Field Hybrid Switching in Maize (Zea mays L.)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 15
Issue 3
10.3390/agronomy15030719
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effects and Mechanism of Auxin and Its Inhibitors on Root Growth and Mineral Nutrient Absorption in Citrus (Trifoliate Orange, Poncirus trifoliata) Seedlings via Its Synthesis and Transport Pathways

by
Yuwei Yang
1,†,
Yidong Shi
1,†,
Cuiling Tong
2 and
Dejian Zhang
1,*
1
College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China
2
Jingzhou Institute of Technology, Jingzhou 434025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(3), 719; https://doi.org/10.3390/agronomy15030719
Submission received: 13 February 2025 / Revised: 8 March 2025 / Accepted: 14 March 2025 / Published: 16 March 2025
(This article belongs to the Topic Plants Nutrients, 2nd Volume)
Browse Figures
Versions Notes

Abstract

:
As an endogenous hormone, auxin plays a crucial role in regulating plants’ growth and development, and also in the responses to abiotic stresses. However, the effects and mechanism of auxin and its inhibitors on plant growth and mineral nutrient absorption in citrus have not been thoroughly studied. Therefore, we used trifoliate orange (citrus’s rootstock, Poncirus trifoliata) as the experimental material to supplement the research content in this area. The trifoliate orange seedlings were treated with exogenous auxin (indolebutyric acid, IBA) and auxin inhibitor (2-naphthoxyacetic acid, 2-NOA) in a sand culture system. The results showed that compared to the control, exogenous auxin (1.0 µmol L−1 IBA) significantly enhanced the taproot length, lateral root length, and lateral root number by 17.56%, 123.07%, and 88.89%, respectively, while also markedly elevating the levels of nitrogen (N), phosphorus (P), potassium (K), copper (Cu), and zinc (Zn) by 14.29%, 45.61%, 23.28%, 42.86%, and 59.80%, respectively. Again compared to the control, the auxin inhibitor (50.0 µmol L−1 2-NOA) dramatically reduced the taproot length, lateral root length, and lateral root number by 21.37%, 10.25%, and 43.33%, respectively, while also markedly decreasing the levels of N, magnesium (Mg), iron (Fe), Cu, and Zn by 7.94%, 10.42%, 24.65%, 39.25%, and 18.76%, respectively. Furthermore, IBA increased auxin accumulation in the root hair, stele, and epidermal tissues of citrus taproots, and promoted the up-regulation of auxin synthesis genes (TAR2, YUC3, YUC4, YUC6, YUC8) and transport genes (ABCB1, ABCB19, AUX1, LAX1, LAX2, PIN1, PIN3, PIN4). In contrast, 2-NOA decreased auxin levels in the root hair, stele, and epidermal tissues of citrus taproots, and was involved in the down-regulation of auxin synthesis genes (TAR2, YUC3, YUC4, YUC6) and transport genes (ABCB1, AUX1, LAX1, LAX2, LAX3, PIN3). Interestingly, 2-NOA dramatically elevated auxin level specifically in the root tip of citrus taproot. Therefore, 2-NOA disrupts auxin reflux from the root tip to root hair and epidermal tissues in citrus taproot through down-regulation of auxin transport genes, thereby creating localized (i.e., root hair zone and epidermal tissues) auxin deficiencies that compromise root system architecture and nutrient acquisition capacity. According to the results of this study, exogenous auxin analogs could regulate citrus growth and mineral nutrient absorption through the auxin synthesis and transport pathways.

1. Introduction

During plant growth and development, auxin is involved at nearly every stage, from seed germination to aging and death. Auxin biosynthesis pathways are primarily classified into tryptophan (Trp)-dependent and Trp-independent mechanisms. The predominant routes of auxin production in plants occur through Trp-dependent pathways, which encompass four major enzymatic routes: the indole-3-acetamide (IAM) pathway, indole-3-pyruvate (IPA) pathway, tryptamine (TAM) pathway, and the indole-3-acetaldoxime (IAOx) pathway [1]. The auxin synthesis and transport pathway is an extremely complex system that plays a crucial role in plant growth and development and their responses to stress [2,3]. However, current research on the regulation of plant roots through signaling pathways remains incomplete.
The citrus fruit, renowned for its distinctive flavor and abundant nutritional content, is extensively cultivated in 140 countries worldwide, making it the most widely produced fruit globally [4]. However, soil erosion leads to the loss of organic matter, which in turn reduces the water and nutrient retention capacity of the soil, thus adversely affecting citrus production [5]. Therefore, understanding the effects of auxin on citrus roots is crucial to enhancing the mineral element absorption level and stress resistance of citrus plants.
In general, roots are mainly divided into two types: primary roots formed during embryo development and secondary roots formed after embryo development [6]. These secondary roots include lateral roots (LRs), which branch from the main root, and adventitial roots (ARs), which develop on non-root tissues such as hypocotyls, stems, and leaves [7]. Auxin, as the main plant hormone, plays an important role in root development. Its ability to drive root growth and development in response to the regulatory networks of related genes has been well characterized [8].
Auxin synthesis genes, as well as input and output vectors, play an important role in auxin signal transmission. The regulation of the plant root structure mainly depends on the active auxin-synthesizing genes tryptophan aminotransferase-related (TAR) and yucca (YUC) in the apex, which induce cells to produce auxin through specific signaling pathways [9,10]. AUX1 plays an important role in regulating the asymmetric distribution of auxin in root tips, especially in synergistic action with LIKE AUX3 (LAX3) to regulate plant auxin levels [11,12]. The directed flow of auxin between cells is closely related to PIN efflux transporters. In the PIN gene family, PIN1 is a target regulating auxin action, while PIN3 is an auxin response factor in ARF7-mediated lateral root development [12,13]. In addition, SWI/SNF chromatin also promotes root stem cell niche formation by targeting PIN4 [13]. As a novel vector, the ABCB protein family plays a regulatory role in maintaining auxin levels during plant tissue mitosis [14].
Mineral nutrients are selectively absorbed through the root system to regulate plant growth and development [15,16]. Mineral elements play a variety of important roles in plants. For example, phosphorus (P) and potassium (K) regulate the sugar metabolism pathway of the plant, while magnesium (Mg) influences photosynthesis and respiration [17,18]. It is worth noting that zinc (Zn) has a very important relationship with the synthesis and transport of auxin [19].
Although the effects of auxin on plant growth and mineral nutrient absorption in plants have been thoroughly studied, the effects of auxin and its inhibitors on root growth and mineral nutrient absorption in citrus have not been entirely elucidated. For example, it is not clear whether auxin and its inhibitors regulate citrus root growth and nutrient absorption through auxin synthesis and transport. Therefore, we conducted this study to explore the regulatory mechanisms of IBA and 2-NOA on the root growth and mineral nutrient absorption of citrus seedlings. By analyzing the effects of exogenous treatment on taproot elongation, lateral root density, mineral element content, and related gene expression, the key role of auxin in root development and nutrient absorption was clarified. This study further revealed the internal molecular mechanism of auxin’s influence on root structure and function by regulating the expression dynamics of synthesis and transport genes, in order to provide a theoretical basis for optimizing root management and mineral nutrition regulation in citrus cultivation, and to help improve citrus stress resistance and production efficiency.

2. Materials and Methods

2.1. Materials and Growth Conditions

In this study, trifoliate orange (Poncirus trifoliata) seeds purchased from the experimental base of the Wuhan Forest Fruit Promotion Station were selected as experimental materials. First, the river sand used for planting materials was washed three times with tap water and pure water, and then sterilized using a high-temperature autoclave to ensure cleanliness. The high temperature autoclave (121 °C, 15 psi) lasted for 60 min, and the time count began when the center of the log reached 121 °C). The sterilization process was carried out in two high-temperature cycles, with cooling during the cycle interval. Then, full and consistent seeds were selected, soaked, and disinfected with 75% (v/v) ethanol for 15 min, and washed 4–5 times with sterile water. After disinfection, the seeds were placed in a sterilized tray and placed in a dark incubator at 28 °C for germination. When the germinated seeds were 1 cm in length, they were sown in round pots (height × length = 30 cm × 20 cm) filled with sand and placed in a greenhouse.
The transplanted potted seedlings were cultured under 16 h of light (28 °C) and 8 h of darkness (22 °C). Nutrient solution was applied from the day of transplantation and the seedlings were watered every 3 days with a dosage of 100 mL each time. After the growth of three true leaves, the treatment began. All seedlings were irrigated with full Hoagland solution (100 mL) once every 3 days.
The basic culture Hoagland solution was composed of the following: 6.00 mM KNO3, 4.00 mM Ca (NO3)2·4H2O, 1.00 mM NH4H2PO4, 2.00 mM MgSO4·7H2O, 9.40 μM MnCl2·4H2O, 0.34 μM CuSO4·3H2O, 0.74 μM ZnSO4·7H2O, 48.00 μM H3BO3, 0.14 μM H2MoO4, and 50.00 mM EDTA-Fe, at 5.84–6.02 pH.

2.2. Experiment Design

The seedlings were first treated with growth regulators (Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China) on 26 February 2024 in three different ways: the control group (CK), 1.0 µmol −1 IBA, and 50.0 µmol L−1 2-NOA. Four pots were treated, each containing 10 seeds, and the process was repeated three times. Following 10 treatment applications, the test material was removed, and the root morphology was determined. The cultivation substrate was stripped away, and the fine sand on the root surface was washed with water to determine the root morphology. After 15 treatments, the same method was used to remove the test material for subsequent analysis.

2.3. Study of Root Morphology

After 10 treatments of the transplanted test material, 3 seedlings with more consistent growth were selected from the pot seedlings, and the length of the taproot and lateral root, the diameter of the taproot, and the number of lateral roots were measured with a ruler. Each index was measured three times.

2.4. Mineral Nutrient Content Analysis

The test materials of the aboveground and underground parts of each treatment were packaged and dried in an oven at 105 °C to constant weight (about 48 h). After crushing, 0.5 g of the test materials was ground into a powder with liquid nitrogen. After weighing, 0.2 g of the sample was placed into a muffle furnace (500 °C) for ashing for 10 h, then removed and dissolved with 0.1 mol L−1 HCl. The dissolved powder was sent to the Institute of Soil Science, Nanjing, Chinese Academy of Sciences, where the contents of phosphorus, potassium, calcium, magnesium, iron, copper, zinc, boron, and other mineral nutrients were determined using an atomic absorption spectrometer (SPECTR AA220, Varian Co., Palo Alto, CA, USA). The determination method followed that of Borah et al. [20]. The total nitrogen content was determined using a plasma emission spectrometer (ICP Spectrometer, Model: IRIS Advantage, Edison, NJ, USA) and analyzed following Kramberger’s method [21].

2.5. Root Auxin Content

The auxin content of four regions of the taproot was separately quantified using gas chromatography–mass spectrometry (GC-MS): root tips (0–0.5 cm from the apex), root hair zones (2–3 cm from the apex), epidermis (4–5 cm from the apex), and stele (4–5 cm from the apex).
The relevant test materials from each treatment were collected, frozen, and stored in dry ice. These samples were sent to the Engineering Research Center of the Ministry of Education of Plant Growth Regulators, China Agricultural University, for IAA determination. Each treatment was repeated three times, and the determination followed Ali et al.’s method [22].

2.6. Analysis of Gene Expression

Gene selection: Auxin synthesis and transport genes were screened using the Sweet Orange whole genome databases (http://citrus.hzau.edu.cn/index.php, accessed on 10 February 2025) [23].
Primer design and synthesis: Gene primers were designed using Primer 5, and the housekeeping gene β-actin was selected as the internal reference gene to amplify with other target genes and correct differences in their relative expression levels across each sample (Table 1, [24]). The primers were synthesized by BGI Co., Ltd., Shenzhen, China. Before use, the primers were centrifuged at 10,000× g at room temperature for 1 min. RNase-free H2O was added according to the corresponding concentration of the respective primers, diluted to 10 μmol L−1, and the solution was stored in a refrigerator at 4 °C after gentle mixing.
RNA extraction: Root segments 0–1 cm and 2–3 cm away from the lateral root tip were frozen in liquid nitrogen for 1 min and then stored in an ultra-low temperature refrigerator at −80 °C for total RNA extraction. The extraction method followed the product manual of the kit.
Synthesis of first strand cDNA: cDNA was synthesized using PrimeScript@RT reagent Kit (TaKaRa, Chiyoda, Japan) kit, and gDNA was removed using the gDNA Eraser in the kit
Fluorescence quantitative PCR: The relative expression levels of each gene in each sample were determined using the SYBRGreenI incorporation method on a Q7 real-time quantitative PCR instrument (ABI, Los Angeles, CA, USA). The fluorescent quantitative PCR reagent was SYBR@PreMix Ex TaqTM (TaKaRa, Japan). Relative quantitative PCR was calculated using the Livak and Schmittgen [25] 2−ΔΔCT method, and each reaction was repeated four times.

2.7. Statistical Analysis

Data and graphics were processed using Microsoft Excel 2010 and Photoshop 7.0.1 software. All test data were analyzed using SAS mathematical statistics analysis software (version 8.1) to perform an ANOVA for testing significant differences (p < 0.05).

3. Results

3.1. The Morphology of Trifoliate Orange Seedlings

As shown in Figure 1 and Figure 2, IBA and 2-NOA treatments had a significant effect on trifoliate orange seedlings. Although exogenous IBA treatment did not cause significant changes in the aboveground part of the plant, root development in the underground part was noticeably more robust. Compared with the control, the length of the taproot and lateral root density increased significantly after IBA treatment. In contrast, the potted plants treated with 2-NOA were shorter, with sparse and yellow leaves. The root system after 2-NOA treatment was weaker than that of the control group, with shorter taproots and shorter lateral roots.

3.2. The Growth of Main and Lateral Roots

To quantitatively evaluate the effects of auxin and its inhibitors on taproot and lateral root development of trifoliate orange seedlings, we analyzed the root phenotype data of trifoliate orange seedlings in each treatment group. As shown in Figure 1, after IBA treatment, the length and diameter of the taproot significantly increased by 17.56% and 18.18% compared with the control group, respectively. After IBA treatment, the lateral root length and number increased by 123.08% and 88.89%, respectively, compared with the control. In contrast, compared with the control group, 2-NOA treatment reduced the taproot length and diameter by 21.37% and 9.00%, respectively. After 2-NOA treatment, the lateral root length and number were reduced by 10.26% and 88.89%, respectively, compared with the control. These results indicate that auxin promoted the growth of citrus roots, while auxin inhibitors showed obvious inhibition (Table 2).

3.3. Variations in Plant Mineral Nutrient Composition

According to Figure 2, treatment with IBA and its inhibitor 2-NOA had varying effects on the aboveground and root mineral concentrations of trifoliate orange seedlings. Compared with the control, IBA treatment had the greatest effect on the content of P, Cu, and Zn in orange root, which increased by 45.61%, 42.86%, and 59.80%, respectively. Additionally, after IBA treatment, the contents of N, K, and Ca in orange root were increased by 14.29%, 23.28%, and 17.24%, respectively. In aboveground tissues, the effect of IBA treatment on the Zn concentration was 38.14% higher than that of the control. Similarly, after IBA treatment, the concentrations of N, P, K, Mg, Fe, and B in aboveground tissues increased by 14.63%, 20.29%, 16.67%, 6.25%, 22.73%, and 6.49%, respectively, while the concentrations of Ca did not change. In contrast, 2-NOA treatment significantly reduced the Cu content in orange root by 39.29% compared with the control. After 2-NOA treatment, the contents of N, K, Mg, and Fe in orange root decreased by 7.94%, 8.21%, 10.42%, and 24.65%, respectively, while the content of P did not change. In the aboveground tissue, the concentrations of N, P, K, and Mg after 2-NOA treatment decreased by 8.54%, 5.80%, 8.33%, and 7.50%, respectively, compared with the control group. Notably, after 2-NOA treatment, the concentration of Cu and B in aboveground tissues increased by 8.33% and 3.9%, respectively.

3.4. Measurement of Endogenous Auxin Content in Roots

To study the effects of exogenous auxin and its inhibitors on endogenous auxin, we compared the concentrations of endogenous IAA in the tissues of the root tip, root hair, epidermis, and stele of the taproot. Compared with the control group, IBA markedly increased the auxin concentration in the root hair and the epidermis by 23.58% and 88.64%, respectively, while its effect on the root tip and epidermis was relatively minor, increasing the auxin concentration by 6.45% and 7.94%, respectively (Figure 3). Interestingly, after treatment with 2-NOA, the auxin concentration in the root tip increased considerably, by 30.32%. In contrast, the concentrations in the root hair and the stele in 2-NOA treatment were decreased significantly by 18.87% and 24.60%, respectively. Remarkably, the concentrations of endogenous IAA in the taproot epidermis remained largely unaffected while treated by 2-NOA.

3.5. The Expression Levels of Root Auxin Biosynthesis and Transport Genes

Auxin synthesis is regulated by related genes, such as the TAR gene, which regulates the conversion of tryptophan to indole 3-pyruvate (IPA), while the YUCCA family genes primarily promote the conversion of IPA to IAA. It is generally believed that the root tip is a key site for plant auxin synthesis; therefore, the root tip was selected for expression analysis. In this experiment, the relative expression levels of auxin synthesis-related genes (TAR2, YUC3, YUC4, YUC6, YUC8) in lateral root tips were cloned and measured under exogenous auxin and its inhibitor treatments (Figure 4). As shown in Figure 4, TAR2, YUC3, YUC4, YUC6, and YUC8 were positively regulated by IBA, with TAR2 exhibiting the highest expression level, while 2-NOA markedly reduced its relative expression. In contrast, YUC8 was positively regulated by IBA, and 2-NOA treatment had minimal effect on its expression.
Auxin transport occurs through two modes: fast, long-distance, non-specific non-polar transport, and slower polar transport regulated by transport carriers. In this experiment, the relative expression levels of trifoliate orange auxin transport vector genes (AUX1, LAX1, LAX2, LAX3, PIN1, PIN3, PIN4, ABCB1, ABCB19) in lateral root tips were cloned and measured (Figure 5). As shown in Figure 5, IBA treatment strikingly up-regulated the expression levels of AUX1, LAX1, and LAX2 in the input vector, while 2-NOA treatment significantly down-regulated their expression levels. The relative expression of LAX3 was less pronounced than that of AUX1, LAX1, and LAX2; however, 2-NOA treatment still led to down-regulated expression. As shown in Figure 5, IBA treatment markedly up-regulated the expression levels of PIN1, PIN3, and PIN4 in root tips within the output vector. In contrast, 2-NOA treatment observably down-regulated the expression levels of PIN3 in root tips but had no significant effect on PIN1 and PIN4. The effects of the ABCB family on auxin transport in lateral root apex were analyzed by examining the ABCB1 and ABCB19 genes. IBA treatment prominently up-regulated the expression levels of both ABCB1 and ABCB19. However, 2-NOA treatment markedly down-regulated the expression level of ABCB1 but had no significant effect on ABCB19.

4. Discussion

4.1. Effect of Auxin on the Root Growth of Trifoliate Orange

The results showed that IBA promoted the growth and development of the roots of trifoliate orange seedlings. Roychoudhry and Kepinski [26] demonstrated that auxin plays an important role in triggering and supporting plant root development, as well as in molecular regulatory pathways, which is consistent with our results. Previous studies have shown that auxin synthesis, transport, and signaling have significant effects on root growth and development [27]. This was consistent with our observation that exogenous auxin significantly promoted the development of the trifoliate orange root system, increasing taproot length, taproot diameter, lateral root length, and lateral root number to varying degrees. In particular, the lateral root growth was markedly enhanced. In contrast, the results indicated that 2-NBA had an inhibitory effect on root growth, particularly on taproot length and the number of lateral roots. This is likely because 2-NOA affects the transport of auxin from the root tip to the base and the root hair area, leading to a reduced auxin concentration in the root hair area [28].

4.2. Effect of Auxin on Mineral Nutrients of Trifoliate Orange

As a major regulatory factor in root development, auxin can promote root growth, thereby increasing root surface area and mineral absorption [29]. Minerals are important nutrient regulators in plant root development, which can promote taproot elongation and lateral root formation by activating auxin-mediated signaling pathways [30,31,32]. Through the joint regulation of mineral nutrition, root growth and development are further promoted [33,34]. After treating plants with the auxin inhibitor 2-NOA, 2-NOA inhibited root growth by inhibiting auxin inflow, thus affecting nutrient uptake [35,36]. Our results showed that after IBA treatment, the concentrations of N, P, K, Ca, Cu, and Zn in the root system observably increased, while the concentrations of Mg, Fe, and B remained almost unchanged. The two nutrients K and N both play key roles in the photosynthesis process and the subsequent long-distance transport of photoassimilates [37,38]. This means that auxin promotes the absorption of mineral nutrients from the roots of the plant, and indirectly promotes the progress of photosynthesis. This indicated that IBA could enhance the efficiency of mineral nutrient uptake by plant roots. In contrast, 2-NOA significantly decreased the concentrations of N, K, Mg, Fe, and Cu in roots, slightly decreased the concentrations of B and Ca, and had minimal effects on P and Zn. Previous studies have shown that N is an essential component of plant metabolic activities, and P plays a key role in important metabolic processes. Photosynthesis and respiration rely heavily on K and Cu, which play particularly important roles in these processes. Therefore, it can be inferred that auxin enhances the growth capacity of trifoliate orange. However, after treatment with auxin inhibitors, the growth potential of the plants was markedly reduced [39,40,41,42,43,44]. After treatment with IBA, the contents of N, P, K, and Fe in trifoliate orange shoots were significantly increased, while the concentrations of Mg, Cu, Zn, and B slightly increased, with no change observed in the Ca concentration. In the aboveground tissues treated with 2-NOA, the concentrations of N, P, K, and Mg decreased notably, while Fe, Zn, and B showed smaller decreases, and the concentrations of Cu and Ca remained unchanged. The changes in mineral concentrations in the aboveground tissues were generally consistent with those in the root system, except the concentration of Ca, which remained unaffected under any treatment. Based on the research of Peaucelle et al., this stability in Ca concentration may be attributed to its primary role in maintaining cell wall stability, which is not influenced by auxin in aboveground tissues [45].

4.3. Expression of Auxin and Its Related Genes

In this study, the expression of several auxin synthesis genes and transport vector genes in the apex of trifoliate orange roots was analyzed. According to the experimental results, IBA treatment substantially increased the expression levels of auxin synthesis genes, including TAR2, YUC3, YUC4, YUC6, and YUC8. Moreover, the expression levels of auxin transport genes AUX1, LAX1, LAX2, PIN1, PIN3, PIN4, ABCB1, and ABCB19 were also up-regulated. This may be because exogenous auxin treatment significantly improved auxin synthesis and transport in trifoliate orange, consistent with the findings of Du et al. [46] and Leftley et al. [47]. Our study further demonstrated that exogenous auxin can stimulate the expression of PIN, YUC, LAX, and AUX family genes in plants, thereby promoting the growth and development of plants, consistent with previous studies [48,49]. Under 2-NOA treatment, the expression levels of auxin synthesis genes (TAR2, YUC3, YUC4, and YUC6) and auxin transport genes (AUX1, LAX1, LAX2, LAX3, PIN3, and ABCB1) were reduced, whereas the auxin output vectors PIN1 and PIN4 were unaffected. These results align with the findings of Zhang et al. [50] and Xi et al. [51]. PIN1 primarily regulates the polar transport of auxin to the root cap in mid-column tissues, promoting taproot elongation, while PIN4 mainly influences the polar transport of auxin to the root tip, thereby regulating root tip growth [50,51]. Therefore, it is speculated that 2-NOA does not impede auxin transport from the apex to the root tip but inhibits auxin transport from the base to the root hair region by down-regulating the expression of related auxin transport vector genes. This markedly reduces the auxin concentration in the root hair region, thereby inhibiting the normal development of the root hair in this region.

5. Conclusions

In this study, exogenous auxin (IBA) was shown to promote citrus growth, especially the root architecture and nutrient levels, while the auxin inhibitor (2-NOA) produced the opposite effects. Further, the auxin concentrations in different parts of the taproot and the expression of auxin synthesis and transport genes were analyzed. It was found that IBA increased auxin accumulation in the root hair, stele, and epidermal tissues of citrus taproots through the up-regulation of auxin synthesis genes (TAR2, YUC3, YUC4, YUC6, YUC8) and transport genes (ABCB1, ABCB19, AUX1, LAX1, LAX2, PIN1, PIN3, PIN4). In contrast, 2-NOA decreased auxin levels in the root hair, stele, and epidermal tissues of citrus taproots through the down-regulation of auxin synthesis genes (TAR2, YUC3, YUC4, YUC6) and transport genes (ABCB1, AUX1, LAX1, LAX2, LAX3, PIN3). Interestingly, 2-NOA dramatically and specifically elevates the auxin levels in the root tip of the citrus taproot. These findings suggest that 2-NOA disrupts auxin reflux from the root tip to the root hair and epidermal tissues in the citrus taproot through the down-regulation of auxin transport genes, thereby creating localized (i.e., the root hair zone and epidermis) auxin deficiencies that compromise the root system architecture and nutrient acquisition capacity.

Author Contributions

Y.Y. and Y.S.; methodology, Y.Y.; software, Y.S.; validation, C.T. and D.Z.; formal analysis, C.T.; investigation, Y.Y. and Y.S.; resources, D.Z.; data curation, Y.Y. and Y.S.; writing—original draft preparation, Y.Y. and Y.S.; writing—review and editing, C.T. and D.Z.; visualization, C.T.; supervision, C.T.; project administration, D.Z.; funding acquisition, D.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32001984).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Zhao, Y.D. Auxin biosynthesis and its role in plant development. Annu. Rev. Plant Biol. 2010, 61, 49–64. [Google Scholar] [CrossRef]
  2. Cavallari, N.; Artner, C.; Benkova, E. Auxin-regulated lateral root organogenesis. Csh Perspect. Biol. 2021, 13, a039941. [Google Scholar] [CrossRef] [PubMed]
  3. Lavy, M.; Estelle, M. Mechanisms of auxin signaling. Development 2016, 143, 3226–3229. [Google Scholar] [CrossRef] [PubMed]
  4. Chen, Y.; Barzee, T.J.; Zhang, R. Citrus: Integrated Processing Technologies for Food and Agricultural by-Products; Academic Press: Cambridge, MA, USA, 2019; pp. 217–242. [Google Scholar] [CrossRef]
  5. Eekhout, J.P.C.; de Vente, J. Global impact of climate change on soil erosion and potential for adaptation through soil conservation. Earth-Sci. Rev. 2022, 226, 103921. [Google Scholar] [CrossRef]
  6. Rivas, M.Á.; Friero, I.; Alarcón, M.V.; Salguero, J. Auxin-cytokinin balance shapes maize root architecture by controlling primary root elongation and lateral root development. Front. Plant Sci. 2022, 13, 836592. [Google Scholar] [CrossRef]
  7. Qin, H.; Huang, R. Auxin controlled by ethylene steers root development. Int. J. Mol. Sci. 2018, 19, 3656. [Google Scholar] [CrossRef] [PubMed]
  8. Jeon, B.W.; Kim, M.-J.; Pandey, S.K.; Oh, E.; Seo, P.J.; Kim, J. Recent advances in peptide signaling during Arabidopsis root development. J. Exp. Bot. 2021, 72, 2889–2902. [Google Scholar] [CrossRef]
  9. Thelander, M.; Landberg, K.; Muller, A.; Cloarec, G.; Cunniffe, N.; Huguet, S.; Soubigou-Taconnat, L.; Brunaud, V.; Coudert, Y. Apical dominance control by TAR-YUC-mediated auxin biosynthesis is a deep homology of land plants. Curr. Biol. 2022, 32, 3838–3846.e5. [Google Scholar] [CrossRef]
  10. Kim, H.; Jang, J.; Seomun, S.; Yoon, Y.; Jang, G. Division of cortical cells is regulated by auxin in Arabidopsis roots. Front. Plant Sci. 2022, 13, 953225. [Google Scholar] [CrossRef]
  11. Swarup, R.; Péret, B. AUX/LAX family of auxin influx carriers—An overview. Front. Plant Sci. 2012, 3, 32237. [Google Scholar] [CrossRef]
  12. Singh, G.; Retzer, K.; Vosolsobě, S.; Napier, R. Advances in understanding the mechanism of action of the auxin permease AUX1. Int. J. Mol. Sci. 2018, 19, 3391. [Google Scholar] [CrossRef] [PubMed]
  13. Zhou, J.J.; Luo, J. The PIN-FORMED auxin efflux carriers in plants. Int. J. Mol. Sci. 2018, 19, 2759. [Google Scholar] [CrossRef] [PubMed]
  14. Vosolsobě, S.; Skokan, R.; Petrášek, J. The evolutionary origins of auxin transport: What we know and what we need to know. J. Exp. Bot. 2020, 71, 3287–3295. [Google Scholar] [CrossRef]
  15. Fageria, N.K.; Moreira, A. The role of mineral nutrition on root growth of crop plants. Adv. Agron. 2011, 110, 251–331. [Google Scholar] [CrossRef]
  16. Pandey, R. Mineral nutrition of plants. Photosynth. Res. 2015, 46, 499–538. [Google Scholar] [CrossRef]
  17. Sardans, J.; Peñuelas, J. Potassium control of plant functions: Ecological and agricultural implications. Plants. 2021, 10, 419. [Google Scholar] [CrossRef]
  18. Farhat, N.; Elkhouni, A.; Zorrig, W.; Smaoui, A.; Abdelly, C.; Rabhi, M. Effects of magnesium deficiency on photosynthesis and carbohydrate partitioning. Acta Physiol. Plant. 2016, 38, 145. [Google Scholar] [CrossRef]
  19. Wang, J.; Yang, S. Dose-dependent responses of Arabidopsis thaliana to zinc are mediated by auxin homeostasis and transport. Environ. Exp. Bot. 2021, 189, 104554. [Google Scholar] [CrossRef]
  20. Borah, S.; Baruah, A.M.; Das, A.K.; Borah, J. Determination of mineral content in commonly consumed leafy vegetables. Food Anal. Method. 2009, 2, 226–230. [Google Scholar] [CrossRef]
  21. Kramberger, B.; Gselman, A.; Janzekovic, M.; Kaligaric, M.; Bracko, B. Effects of cover crops on soil mineral nitrogen and on the yield and nitrogen content of maize. Eur. J. Agron. 2009, 31, 103–109. [Google Scholar] [CrossRef]
  22. Ali, B.; Sabri, A.; Ljung, K.; Hasnain, S. Auxin production by plant associated bacteria: Impact on endogenous IAA content and growth of Triticum aestivum L. Lett. Appl. Microbiol. 2009, 48, 542–547. [Google Scholar] [CrossRef] [PubMed]
  23. Xu, Q.; Chen, L.-L.; Ruan, X.; Chen, D.; Zhu, A.; Chen, C.; Bertrand, D.; Jiao, W.-B.; Hao, B.-H.; Lyon, M.P.; et al. The draft genome of sweet orange (Citrus sinensis). Nat. Genet. 2013, 45, 59–66. [Google Scholar] [CrossRef] [PubMed]
  24. Hu, C.-H.; Yuan, S.-D.; Tong, C.-L.; Zhang, D.-J.; Huang, R.-H. Ethylene modulates root growth and mineral nutrients levels in trifoliate orange through the auxin-signaling pathway. Not. Bot. Horti Agrobo. 2023, 51, 13269. [Google Scholar] [CrossRef]
  25. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  26. Roychoudhry, S.; Kepinski, S. Auxin in root development. CSH Perspect. Biol. 2022, 14, a039933. [Google Scholar] [CrossRef]
  27. Robert, H.S.; Friml, J. Auxin and other signals on the move in plants. Nat. Chem. Biol. 2009, 5, 325–332. [Google Scholar] [CrossRef]
  28. Saini, S.; Sharma, I.; Kaur, N.; Pati, P.K. Auxin: A master regulator in plant root development. Plant Cell Rep. 2013, 32, 741–757. [Google Scholar] [CrossRef]
  29. Bouain, N.; Krouk, G.; Lacombe, B.; Rouached, H. Getting to the root of plant mineral nutrition: Combinatorial nutrient stresses reveal emergent properties. Trends Plant Sci. 2019, 24, 542–552. [Google Scholar] [CrossRef]
  30. Chen, J.; Liu, L.; Wang, Z.; Zhang, Y.; Sun, H.; Song, S.; Bai, Z.; Lu, Z.; Li, C. Nitrogen fertilization increases root growth and coordinates the root–shoot relationship in cotton. Front. Plant Sci. 2020, 11, 880. [Google Scholar] [CrossRef]
  31. Xu, Q.; Fu, H.; Zhu, B.; Hussain, H.A.; Zhang, K.; Tian, X.; Duan, M.; Xie, X.; Wang, L. Potassium improves drought stress tolerance in plants by affecting root morphology, root exudates, and microbial diversity. Metabolites 2021, 11, 131. [Google Scholar] [CrossRef]
  32. Omondi, J.O.; Lazarovitch, N.; Rachmilevitch, S.; Yermiyahu, U. Phosphorus affects storage root yield of cassava through root numbers. J. Plant Nut. 2019, 42, 2070–2079. [Google Scholar] [CrossRef]
  33. Salas-González, I.; Reyt, G.; Flis, P.; Custódio, V.; Gopaulchan, D.; Bakhoum, N.; Dew, T.P.; Suresh, K.; Franke, R.B.; Dangl, J.L.; et al. Coordination between microbiota and root endodermis supports plant mineral nutrient homeostasis. Science 2021, 371, eabd0695. [Google Scholar] [CrossRef] [PubMed]
  34. Phuphong, P.; Cakmak, I.; Yazici, A.; Rerkasem, B.; Prom-U-Thai, C. Shoot and root growth of rice seedlings as affected by soil and foliar zinc applications. J. Plant Nut. 2020, 43, 1259–1267. [Google Scholar] [CrossRef]
  35. Laňková, M.; Smith, R.S.; Pešek, B.; Kubeš, M.; Zažímalová, E.; Petrášek, J.; Hoyerová, K. Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells. J. Exp. Bot. 2010, 61, 3589–3598. [Google Scholar] [CrossRef]
  36. Zhang, D.-J.; Yang, Y.-J.; Liu, C.-Y.; Zhang, F.; Hu, W.; Gong, S.-B.; Wu, Q.-S. Auxin modulates root-hair growth through its signaling pathway in citrus. Sci. Hortic. 2018, 236, 73–78. [Google Scholar] [CrossRef]
  37. Tränkner, M.; Tavakol, E.; Jákli, B. Functioning of potassium and magnesium in photosynthesis, photosynthate translocation and photoprotection. Physiol. Plant. 2018, 163, 414–431. [Google Scholar] [CrossRef] [PubMed]
  38. Mu, X.; Chen, Y. The physiological response of photosynthesis to nitrogen deficiency. Plant Physiol. Biochem. 2021, 158, 76–82. [Google Scholar] [CrossRef]
  39. Ye, X.; Chen, X.-F.; Deng, C.-L.; Yang, L.-T.; Lai, N.-W.; Guo, J.-X.; Chen, L.-S. Magnesium-deficiency effects on pigments, photosynthesis and photosynthetic electron transport of leaves, and nutrients of leaf blades and veins in Citrus sinensis seedlings. Plants 2019, 8, 389. [Google Scholar] [CrossRef]
  40. Adil, M.; Bashir, S.; Bashir, S.; Aslam, Z.; Ahmad, N.; Younas, T.; Asghar, R.M.A.; Alkahtani, J.; Dwiningsih, Y.; Elshikh, M.S. Zinc oxide nanoparticles improved chlorophyll contents, physical parameters, and wheat yield under salt stress. Front. Plant Sci. 2022, 13, 932861. [Google Scholar] [CrossRef]
  41. Li, X.; Kang, H.; Chen, S.; Bai, M.; Li, F.; Liu, T.; Zhou, W.; Aladejana, J.T.; Li, J. Gln-Lys isopeptide bond and boroxine synergy to develop strong, anti-mildew and low-cost soy protein adhesives. J. Clean. Prod. 2023, 397, 136505. [Google Scholar] [CrossRef]
  42. Yang, X.M.; Ren, G.M.; Luo, K.J. Effects of Sepiolite and Mycorrhizals on Nutrient Element Absorptions of Maize in Heavy Metals Contaminated Soil. Adv. Mater. Res. 2013, 800, 153–158. [Google Scholar] [CrossRef]
  43. Jiang, H.-B.; Lu, X.-H.; Deng, B.; Liu, L.-M.; Qiu, B.-S. Adaptive mechanisms of the model photosynthetic organisms, cyanobacteria, to iron deficiency. In Microbial Photosynthesis; Wang, Q., Ed.; Springer: Singapore, 2020; pp. 197–244. [Google Scholar] [CrossRef]
  44. Rawat, J.; Pandey, N.; Saxena, J. Role of potassium in plant photosynthesis, transport, growth and yield. In Role of Potassium in Abiotic Stress; Springer: Berlin/Heidelberg, Germany, 2022; pp. 1–14. [Google Scholar] [CrossRef]
  45. Peaucelle, A.; Braybrook, S.; Höfte, H. Cell wall mechanics and growth control in plants: The role of pectins revisited. Front. Plant Sci. 2012, 3, 121. [Google Scholar] [CrossRef] [PubMed]
  46. Du, Y.; Scheres, B. Lateral root formation and the multiple roles of auxin. J. Exp. Bot. 2018, 69, 155–167. [Google Scholar] [CrossRef] [PubMed]
  47. Leftley, N.; Banda, J.; Pandey, B.; Bennett, M.; Voß, U. Uncovering how auxin optimizes root systems architecture in response to environmental stresses. Csh Perspect. Biol. 2021, 13, a040014. [Google Scholar] [CrossRef]
  48. Habets, M.E.J.; Offringa, R. PIN-driven polar auxin transport in plant developmental plasticity: A key target for environmental and endogenous signals. New Phytol. 2014, 203, 362–377. [Google Scholar] [CrossRef]
  49. Della Rovere, F.; Fattorini, L.; D’Angeli, S.; Veloccia, A.; Falasca, G.; Altamura, M.M. Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of Arabidopsis. Ann. Bot. 2013, 112, 1395–1407. [Google Scholar] [CrossRef]
  50. Zhang, P.; Sun, L.; Qin, J.; Wan, J.; Wang, R.; Li, S.; Xu, J. cGMP is involved in Zn tolerance through the modulation of auxin redistribution in root tips. Environ. Exp. Bot. 2018, 147, 22–30. [Google Scholar] [CrossRef]
  51. Xi, D.; Chen, X.; Wang, Y.; Zhong, R.; He, J.; Shen, J.; Ming, F. Arabidopsis ANAC092 regulates auxin-mediated root development by binding to the ARF8 and PIN4 promoters. J. Integr. Plant Biol. 2019, 61, 1015–1031. [Google Scholar] [CrossRef]
Agronomy 15 00719 g001aAgronomy 15 00719 g001b
Figure 1. Depiction of trifoliate orange seedlings subjected to treatment with IBA and 2-NOA. Note: (a,d)—CK, (b,e)—1.0 µmol L−1 IBA, (c,f)—50 µmol L−1 2-NOA; bar = 5.0 cm.
Figure 1. Depiction of trifoliate orange seedlings subjected to treatment with IBA and 2-NOA. Note: (a,d)—CK, (b,e)—1.0 µmol L−1 IBA, (c,f)—50 µmol L−1 2-NOA; bar = 5.0 cm.
Agronomy 15 00719 g001aAgronomy 15 00719 g001b
Agronomy 15 00719 g002
Figure 2. Effects of IBA and 2-NOA on the concentrations of N, P, K, Ca, Mg, Fe, Cu, Zn, and B in trifoliate orange seedlings. Different letters in the column indicate significant (p < 0.05) differences.
Figure 2. Effects of IBA and 2-NOA on the concentrations of N, P, K, Ca, Mg, Fe, Cu, Zn, and B in trifoliate orange seedlings. Different letters in the column indicate significant (p < 0.05) differences.
Agronomy 15 00719 g002
Agronomy 15 00719 g003
Figure 3. Effects of IBA and 2-NOA on indole-3-acetic acid (IAA) concentrations in the taproot of trifoliate orange. Different letters in the column indicate significant (p < 0.05) differences.
Figure 3. Effects of IBA and 2-NOA on indole-3-acetic acid (IAA) concentrations in the taproot of trifoliate orange. Different letters in the column indicate significant (p < 0.05) differences.
Agronomy 15 00719 g003
Agronomy 15 00719 g004
Figure 4. Relative expression of auxin biosynthesis genes in the root hair zone of the lateral roots in trifoliate orange. Different letters in the column indicate significant (p < 0.05) differences.
Figure 4. Relative expression of auxin biosynthesis genes in the root hair zone of the lateral roots in trifoliate orange. Different letters in the column indicate significant (p < 0.05) differences.
Agronomy 15 00719 g004
Agronomy 15 00719 g005
Figure 5. Relative expression levels of auxin transport genes in the root hair zone of the lateral roots in trifoliate orange. Different letters in the column indicate significant (p < 0.05) differences.
Figure 5. Relative expression levels of auxin transport genes in the root hair zone of the lateral roots in trifoliate orange. Different letters in the column indicate significant (p < 0.05) differences.
Agronomy 15 00719 g005
Table 1. Compilation of the gene-specific primer sequences employed in this study for qRT-PCR.
Table 1. Compilation of the gene-specific primer sequences employed in this study for qRT-PCR.
GeneAccession No.Forward Primer (5′-3′)Reverse Primer (5′-3′)Amplification Size (bp)
ABCB1Ciclev10010916mGAGCCATTCACGCCACTTCTCTTGTAACCGAGCCTTTGAGC186
ABCB19Ciclev10010931mGCATGAGTTTGGGTCAGTCTTTCATCTTCCATTTGTTGGGTCTT127
AUX1Ciclev10011596mCTTGACTCTGCCCTATTCATTCTCTGGACCCAGTAACCCATCAAGC205
LAX1Ciclev10031413mTTGGCGGACATGCAGTGACCAGCGGCAGCAGAAGGAAT123
LAX2Ciclev10028271mTGTGGGAAGATGGGTAGGGACTAGTCATGCTCGCCCACCC98
LAX3Ciclev10001072mATCACTTTCGCTCCTGCTGCCAAACCCAAATCCCACCACTA133
PIN1Ciclev10007787mGCTTTGGCAACAGAAGAGGATTATTACACTTGTCGGCGGCATA94
PIN3orange1.1g006199mCATGCCTCCAGCGAGTGTTATTGCCACCTGAAAGCGATTAGA126
PIN4Ciclev10012938mATGGGGTTGAAAACGAAGGGCCTGATAAGTTTCCTCCACACCA167
TAR2Ciclev10020085mCACACACGGCACACCCCTAGCCTCCCACTCCCCAGATC137
YUC3Ciclev10006828mCCTTCAGGTTTAGCCGTTGCGGAAGTTTGGAAGTTGGCAGA157
YUC4Ciclev10008466mGACCATCTGGGTTAGCCGTTTGTATTTTGGGAAGTTTTCAGGGA185
YUC6Ciclev10008473mGTGGTTGCTAAAGTGGCTGCGTTGAAGGGGACCCAAAAGA122
YUC8Ciclev10020503mGTGATAATGGTAGGGGCAGGAGAATGGCAGGTGAGGGAGC183
β-actinCiclev10025866mCCGACCGTATGAGCAAGGAAATTCCTGTGGACAATGGATGGA190
Table 2. Effects of IBA and 2-NOA on the growth of the tap and lateral roots of trifoliate orange (mean ± SD).
Table 2. Effects of IBA and 2-NOA on the growth of the tap and lateral roots of trifoliate orange (mean ± SD).
TreatmentTap Root Length (cm)Tap Root Diameter (cm)Lateral Root Length (cm)Lateral Root Number (#)
CK5.24 ± 1.48 b0.11 ± 0.01 b0.39 ± 0.08 b1.80 ± 0.44 b
1.0 µmol·L−1 IBA6.16 ± 0.39 a0.13 ± 0.01 a0.87 ± 0.22 a 3.40 ± 1.63 a
50 µmol·L−1 2-NOA4.12 ± 0.14 c0.10 ± 0.01 b0.35 ± 0.03 c1.02 ± 0.10 c
Note: # represents the number of lateral roots. Data (means ± SD, n = 4) followed by different letters in the column indicate significant (p < 0.05) differences—the same as below.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yang, Y.; Shi, Y.; Tong, C.; Zhang, D. Effects and Mechanism of Auxin and Its Inhibitors on Root Growth and Mineral Nutrient Absorption in Citrus (Trifoliate Orange, Poncirus trifoliata) Seedlings via Its Synthesis and Transport Pathways. Agronomy 2025, 15, 719. https://doi.org/10.3390/agronomy15030719

AMA Style

Yang Y, Shi Y, Tong C, Zhang D. Effects and Mechanism of Auxin and Its Inhibitors on Root Growth and Mineral Nutrient Absorption in Citrus (Trifoliate Orange, Poncirus trifoliata) Seedlings via Its Synthesis and Transport Pathways. Agronomy. 2025; 15(3):719. https://doi.org/10.3390/agronomy15030719

Chicago/Turabian Style

Yang, Yuwei, Yidong Shi, Cuiling Tong, and Dejian Zhang. 2025. "Effects and Mechanism of Auxin and Its Inhibitors on Root Growth and Mineral Nutrient Absorption in Citrus (Trifoliate Orange, Poncirus trifoliata) Seedlings via Its Synthesis and Transport Pathways" Agronomy 15, no. 3: 719. https://doi.org/10.3390/agronomy15030719

APA Style

Yang, Y., Shi, Y., Tong, C., & Zhang, D. (2025). Effects and Mechanism of Auxin and Its Inhibitors on Root Growth and Mineral Nutrient Absorption in Citrus (Trifoliate Orange, Poncirus trifoliata) Seedlings via Its Synthesis and Transport Pathways. Agronomy, 15(3), 719. https://doi.org/10.3390/agronomy15030719

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop