- Open Access
Auxin efflux carrier ZmPIN1a modulates auxin reallocation involved in nitrate-mediated root formation
BMC Plant Biology volume 23, Article number: 74 (2023)
Auxin plays a crucial role in nitrate (NO3–)-mediated root architecture, and it is still unclear that if NO3– supply modulates auxin reallocation for regulating root formation in maize (Zea mays L.). This study was conducted to investigate the role of auxin efflux carrier ZmPIN1a in the root formation in response to NO3– supply.
Low NO3– (LN) promoted primary root (PR) elongation, while repressed the development of lateral root primordia (LRP) and total root length. LN modulated auxin levels and polar transport and regulated the expression of auxin-responsive and -signaling genes in roots. Moreover, LN up-regulated the expression level of ZmPIN1a, and overexpression of ZmPIN1a enhanced IAA efflux and accumulation in PR tip, while repressed IAA accumulation in LRP initiation zone, which consequently induced LN-mediated PR elongation and LR inhibition. The inhibition rate of PR length, LRP density and number of ZmPIN1a-OE plants was higher than that of wild-type plants after auxin transport inhibitor NPA treatment under NN and LN conditions, and the degree of inhibition of root growth in ZmPIN1a-OE plants was more obvious under LN condition.
These findings suggest that ZmPIN1a was involved in modulating auxin levels and transport to alter NO3–-mediated root formation in maize.
Nitrogen (N), a critical growth limiting nutrient, is an essential component of several biomolecules and plays a vital role in plant growth and development . Besides, N fertilizers are widely used to ensure higher crop yields. In order to optimize N capture, plants often regularize root architecture to balance N status of the plant and soil . Meanwhile, the heterogeneous reallocation and availability of N nutrients within the soil lead to the development of morphological and physiological plasticity of root architecture with specific functions [3, 4]. Among the various forms of N fertilizers, nitrate (NO3–) is the major form of available inorganic N utilized by most of the crop plants . It has been demonstrated that the localized supply of NO3– modulates the root length and lateral root (LR) formation [5, 6].
Apart from its nutritive role, NO3– also acts as a signaling molecule modulating the root morphology to allow the plants to efficiently adapt to N supply [7, 8]. In Arabidopsis, NO3– has a dual effect on the LR development depending on the concentration and localized supply of NO3–. For examples, low homogeneous NO3– supply promotes LR elongation [9, 10], while represses the LR emergence . Under heterogeneous N supply, high NO3– stimulates the LR growth, but low NO3– represses LR meristem activation [9, 11]. Unlike Arabidopsis, low NO3– supply promotes the elongation of primary root (PR) and LR in maize, while reduces the density of lateral root primordia (LRP), which leads to decline in the overall length of LR [8, 12]. Similarly, N deficiency induces the root elongation and inhibit LR development in rice and wheat [13–15]. Although the knowledge of the NO3–-mediated root architecture is much documented, the understanding of the underlying integrated molecular and physiological mechanism remains inadequate.
Many studies have demonstrated that NO3– stimulates the biosynthesis, polar transport, and signal transduction of auxin, which plays a vital role in the regulation of root development [16, 17]. Localized NO3– supply does not induce the LR elongation in the auxin-insensitive mutant Ataxr4, which indicates a cross-talk between the auxin and NO3– signaling pathways involved in NO3–-mediated root characteristics . Besides, it has also been demonstrated that auxin biosynthesis gene AtTAR2 is required for LR emergence in low NO3–-mediated root formation . Meanwhile, low local NO3– supply reduces auxin concentration in the phloem poles of the LR initiation zone led to less LRP, while enhances auxin accumulation in the root tip for longer roots [12, 17]. These suggested that NO3–-mediated root architecture relies on the distribution and accumulation of auxin in response to N supply.
It is well known that auxin polar transport plays a potential role in root development, which regulates auxin translocation to modulate the LR initiation and elongation [19, 20]. In Arabidopsis, there are eight AtPINs family members, which encode integral membrane proteins with conserved hydrophobic membrane-spanning domains and a less conserved central hydrophilic loop of variable length [21, 22]. In maize, there are eleven PINs genes, there members cluster within the AtPIN1 subclade, namely ZmPIN1a, ZmPIN1b, and ZmPIN1c. The ZmPIN1 proteins showed a typical structure similar to AtPIN1 [21–23]. AtPIN1 mainly resides at the basal end of the vascular cells, and pin1 mutant display a slight reduction of root length and root meristem size . In addition, OsPIN1 and OsPIN2 participate in auxin-dependent adventitious root emergence and LR formation [25, 26]. Correspondingly, the transcription of most auxin efflux carrier (e.g., AtPIN1, AtPIN2, AtPIN4, and AtPIN7) is controlled by NO3– supply in Arabidopsis [27–29]. Localized partial NO3– supply enhances the expression of auxin influx (OsAUX1 and OsAXR4) and efflux (OsPIN1c, OsPIN2, and OsPIN5b) carriers in rice, forming a robust auxin flux to LR zone for enhancing LR initiation [30, 31]. Besides, low NO3– supply promotes root elongation and inhibits LR development via reduced auxin accumulation in the roots by down-regulation of OsPINs . Recently, it has also been reported that OsPIN1b is involved in auxin transport to regulate root apical meristem activity for modulating the elongation of seminal roots under low NO3– condition . Taken together, these results demonstrate that polar auxin transport plays a vital role in NO3–-mediated root architecture.
Maize is a worldwide stable crop with the highest productivity among all crops; besides, it also demands the largest quantity of nitrogen-based fertilizers . However, the global N use efficiency of maize is estimated around 25–50% , indicating the loss of more than half of the N fertilizer applied in maize production. Therefore, it is very imperative to explore the mechanisms involved in the root adaptation to N supply for improving N use efficiency in maize. As previously described, NO3– -mediated root growth is regulated by auxin transport and allocation in maize roots subjected by heterogeneous or uniform NO3– supply [12, 17]. Moreover,  documented that high NO3– supply regulates the expression of ZmPIN1a and ZmPIN9 involved in modulating early pericycle cell division and LR formation in shoot-borne roots by RNA-seq analysis. Moreover, ZmPIN1a overexpression increases auxin transport from shoot to root, and enhances root growth in maize . However, it is yet to be determined whether ZmPIN1a modulate NO3– -mediated root growth by regulating auxin polar transport and localization in maize.
This study was aimed at investigating the role of auxin efflux carrier ZmPIN1a in the development of PR and LR in response to NO3– supply. There had been identified variations in auxin accumulation and reallocation under NN and LN conditions via Non-invasive Micro-test Technology (NMT), DR5rev::RFP marker line, and HPLC–ESI–MS/MS. Meanwhile, we used ZmPIN1a overexpressing transgenic lines, combined with exogenous addition of auxin transport inhibitor NPA to explore the potential role of ZmPIN1a in the NO3–-mediated root architecture. Hence, this study further demonstrated that ZmPIN1a was involved in auxin levels and transport to alter NO3–-mediated root formation in maize.
LN supply enhanced the PR elongation and repressed the LR development
Compared to NN, LN significantly inhibited the shoot growth while intensely promoted primary root elongation (Fig. 1A-B), and the root to shoot ratio was increased by respectively 52% and 70% at 3 and 5 d after LN treatment (Fig. 1C). Moreover, the PR length was increased by respectively 26% and 31% at 3 and 5 d after LN treatment (Fig. 1D). LN treatment repressed the development of LRP (Fig. 1E), and the number of LRP was reduced by respectively 16.5% and 20% at 3 and 5 d after LN treatment (Fig. 1F). The LRP density decreased by respectively 42% and 55% at 3 and 5 d after LN treatment (Fig. 1G). In addition, the total root length was decreased by respectively 10% and 31% at 3 and 5 d after LN treatment (Table S1). These results suggested LN significantly affected the root morphology in maize.
LN supply modulated auxin levels in roots
In order to evaluate the role of auxin in root formation exposed to different NO3– supply, the IAA fluxes along the root tip were examined by using the NMT technique. As shown in Fig. 2A, the IAA fluxes showed the peak of the efflux in the meristem zone at 400 μM from the root tip, suggesting the higher levels of auxin in the meristem zone, which are consistent with previously published results on rice . However, in the LRP initiation zone (500 to 1500 μM from root tip), the IAA fluxes were negative, which indicated a net IAA influx, suggesting the weak auxin activities in this region (Fig. 2A). Meanwhile, LN-treated plants had higher IAA flux values than NN-treated plants at 100, 200, and 400 μM from the root tip, while lower IAA flux values at 700, 900, and 1500 μM from the root tip. In addition, the mean IAA efflux in LN-treated plants was higher by 2.1 folds than that in NN-treated plants at 400 μM from the root tip (Fig. 2B). Inversely, the mean IAA influx in LN-treated plants was higher by 5.3 folds compared to NN-treated plants in the LRP initiation zone (at 1500 μM from the root tip) (Fig. 2C).
To further assess auxin distribution and localization in maize roots responded to NO3– supply, a DR5rev::RFP marker maize line was used to further evaluate IAA status of PR tip and LRP initiation subjected to alter N supply. As shown in Fig. 3A-B, application of naphthylacetic acid (NAA; an analogue of IAA) increased red fluorescent signal, while application of auxin transport inhibitor NPA decreased red fluorescent signal in roots under LN and NN conditions. Compared to NN, red fluorescent signal was stronger in PR tip (Fig. 3A), while weaker in LRP initiation zone under LN condition (Fig. 3B). Meanwhile, IAA levels in the PR tip and LRP initiation zone were measured by using HPLC–MS/MS method. Compared to NN, IAA levels increased by 25% in the PR tip (Fig. 3C), but decreased by 13% in the LRP initiation zone under LN condition (Fig. 3D). These results indicated that LN increased auxin accumulation in PR tip while decreased in the LRP initiation zone.
LN supply regulated the expression of auxin-responsive and -signaling genes in roots
To further confirm the changes of auxin levels induced the response of roots under LN and NN conditions, the transcription level of auxin-responsive genes (ZmIAA2, ZmIAA10, and ZmIAA21) were assessed using RT-qPCR. As expected, the relative expression levels of ZmIAA2, ZmIAA10, and ZmIAA21 were significantly up-regulated in PR tip (Fig. 4A-C), while down-regulated in LRP initiation zone by LN (Fig. 4D-F). The genes (ZmARF7, ZmARF19, and ZmLBD29) involved in LR formation were also evaluated to assess the LRP formation response to LN, and the relative expression of these genes significantly down-regulated by LN in LRP initiation zone (Fig. 4G-I).
In addition, exogenous application of NAA increased the number and density of LRP under LN and NN conditions (Fig. S1). However, application of auxin transport inhibitor NPA decreased the number and density of LRP under LN and NN conditions (Fig. S2).
LN supply altered the expression auxin transport gene ZmPIN1a in roots
Auxin polar transport is critical for accumulation and distribution of auxin in roots, which is carried by auxin influx and efflux transport proteins . Previous study suggested that overexpression of ZmPIN1a increased auxin transport from shoot to root, and promoted root growth in maize . Could ZmPIN1a be involved in modulating root formation under LN condition? The relative expression of ZmPIN1a was examined in the PR tip and LRP initiation zone at different times points after LN treatment. Compared to NN, the relative expression level of ZmPIN1a significantly up-regulated in PR tip (Fig. 5A), similar changes were observed in LRP initiation zone (Fig. 5B), where LN also markedly enhanced the transcript levels of ZmPIN1a. The expression patterns of ZmPIN1a in PR tip and LRP initiation zone were analysed at tissue level using in-situ hybridization. The red hybridization signal of ZmPIN1a was predominantly detected in the meristematic area, epidermis, and central cylinder of the PR tip (Fig. 5C). The expression levels of ZmPIN1a were significantly stimulated by LN treatment in the longitudinal and transverse of PR tip (Fig. 5C), while were also slightly stimulated by LN treatment in the longitudinal and transverse of LRP initiation zone (Fig. 5D). Therefore, ZmPIN1a might be involved in modulating root architecture under LN condition.
Manipulating ZmPIN1a modified root architecture under LN condition
In order to assess the role of ZmPIN1a in modulating root architecture, the transgenic lines overexpressing ZmPIN1a (ZmPIN1a-OE) were evaluated for root phenotypic characteristics under LN and NN conditions. ZmPIN1a-OE plants exhibited higher expression levels of ZmPIN1a (Fig. S3). Overexpression of ZmPIN1a significantly stimulated the PR elongation, while repressed the shoot growth related to the wild-type plants in response to LN and NN treatments (Fig. 6A). In comparison with wild-type plants, the PR length of ZmPIN1a-OE lines (OE 1, OE 3, and OE 5) increased by respectively 13.8, 12.7, and 11.7% under LN condition, but by 7.7, 6.3, and 5.9% under NN condition (Fig. 6B). The LRP density of ZmPIN1a-OE lines decreased by respectively 44.7, 40, and 34.6% under LN condition, but by 28.8, 25.5, and 23.4% under NN condition (Fig. 6C). And similar observations were presented in the number of LRP of ZmPIN1a-OE plants (Fig. 6D). These results suggested ZmPIN1a might be involved in LN-mediated root formation.
Overexpression of ZmPIN1a changed auxin levels and transport in roots subjected to NO3 – supply
To evaluate the role of ZmPIN1a in modulating the auxin reallocation in roots under LN and NN conditions, IAA fluxes were examined in ZmPIN1a-OE roots by using the NMT technique. IAA effluxes in PR tip of ZmPIN1a-OE plants were higher by respectively 73 and 61% than those in wild-type plants under LN condition, but by 60 and 45% under NN condition (Fig. 7A). Meanwhile, IAA influxes in LRP initiation zone of ZmPIN1a-OE plants were higher by respectively 28.4 and 28.1% than those in the wild-type plants under LN condition, but by 23.5 and 17.5% under NN condition (Fig. 7B). The contents of IAA in PR tip and LRP initiation zone were also measured in the ZmPIN1a-OE plants. In comparison with wild-type plants, IAA contents in PR tip of ZmPIN1a-OE plants increased by respectively 19.0 and 17.2% under LN condition, but by 11.7 and 8.5% under NN condition (Fig. 7C). IAA contents in LRP initiation zone of ZmPIN1a-OE plants decreased by respectively 25.0 and 22.8% under LN condition, but by 19.2 and 17.8% under NN condition (Fig. 7D).
In addition, when auxin transport inhibitor NPA was applied under NN condition, the inhibition rate of PR length, LRP density and number of ZmPIN1a-OE plants was higher than that of wild-type plants. More importantly, with the application of NPA under LN condition, the inhibition rate of PR length, LRP density and number of ZmPIN1a-OE plants significantly increased (Fig. 8A-C). These results suggested ZmPIN1a could influence auxin levels and polar transport from shoot to root to alter root formation under LN condition.
Modification in the root architecture has been recognized as an essential adaptive strategy for plants to obtain nutrients from the soil. Previous studies have shown that localized NO3– supply affects the root growth and development in diverse species, which modulate N uptake [9, 17, 30]. Here, uniform LN significantly repressed the LR formation while promoted the PR elongation (Fig. 1), which decreased total root length (Table S1). Although LRs are the major contributors to N uptake, the axial roots could play a more important role for trapping NO3– from the deeper soil under LN than NN .
Many studies have demonstrated that auxin transport and allocation are involved in N-modulated root formation in diverse species [16, 17]. Whether NO3– treatment affects the auxin transport along the roots has yet to be fully uncovered in maize. Here, we provide evidence that auxin transport is crucial for NO3−-mediated root formation. Previous studies have shown that modification of IAA flux affects IAA content, and finally regulate plant development [35, 36]. In this study, the maximal IAA efflux occurred in the meristem zone of the maize root, and similar results are observed in rice , which indicated that higher levels of auxin are present in the meristem zone. Moreover, LN increased the IAA efflux in the meristem zone of root tip, while increased the IAA influx in the LRP initiation zone (Fig. 2). This finding corresponds to the results of uniform LN treatment enhanced the IAA levels in the PR tip, while reduced the IAA levels in LRP initiation zone (Fig. 3C-D). These results demonstrated that NO3− exerts an effect on IAA flux in an indirect manner, which could result in auxin level change in maize root.
In this study, the auxin-responsive marker DR5rev::RFP revealed a more detailed topological view of the effect of LN treatment on the auxin response in PR tip and LRP initiation zone. In the LN-treated plants, the PR tip displayed stronger signal of the auxin-responsive marker DR5rev::RFP (Fig. 3A), responding to the higher IAA effluxes (Fig. 2B) and contents (Fig. 3C), which was consistent with the increased expression of auxin-responsive genes including ZmIAA2, ZmIAA10, and ZmIAA21 (Fig. 4A-C). Similar findings are observed in maize and wheat, where aluminum stress reduces IAA levels and down-regulates the auxin-responsive genes including IAA2, IAA10, and IAA21 in PRs for repressing root elongation [37, 38]. Under LN condition, we found that the expression levels of ZmIAA2, ZmIAA10, and ZmIAA21 were lower (Fig. 4D-F), IAA influxes were higher, IAA contents were lower, and the signal of auxin-responsive marker DR5rev::RFP were weaker in LRP initiation zone than under NN condition. In addition, the LRP initiation is under the control of auxin-responsive transcription factors ARF7 and ARF19, which directly target the downstream auxin-mediated transcription of lateral organ bounda-ries-domain29/asymmetric leaves2-like16 (LBD29) in roots, both of which positively regulate LRP formation [39–41]. Here, the expression of ZmARF7 and ZmARF19, along with their target gene ZmLBD29 were significantly down-regulated by LN treatment in the LRP initiation zone (Fig. 4G-I), consistent with the result of LN inhibiting the LRP formation. In addition, NAA application increased the density of LRP, while NPA treatment reduced the same under LN as well as NN conditions (Fig. S1-2). Similar findings are also observed in previous studies [13, 16, 42]. These results further demonstrated that NO3− exerts an effect on IAA flux in root tips and LRP initiation zone, resulting in a higher IAA content in PR tips, and a longer PR length, a lower IAA content in LRP initiation zones, and a lower LRP density. Consequently, it was inferred that auxin distribution and local concentration variation played important roles to alter auxin signaling for modulating root elongation and LRP formation under different N conditions.
Auxin is known as the focal point of environmental and endogenous signal perception for root formation via regulating auxin polar transport . The polarity of auxin transport is altered by the PIN, AUX1/LAX and ABCB gene families [12, 17, 23, 44] Auxin efflux carrier PIN1 has been identified in Arabidopsis, rice, and maize . The pin1 mutant in both rice and Arabidopsis display a reduction of root length [14, 24]. Similar to the Arabidopsis AtPIN1 orthologue, which controls auxin transport from shoot to root, ZmPIN1a was also found to be involved in regulating plant root development . However, the role of ZmPIN1a in NO3–-mediated root growth was not observed in maize. Here, ZmPIN1a-OE plants were used to investigate the ZmPIN1a on root formation in response to NO3– supply. Our results showed that ZmPIN1a was predominantly expressed in the meristematic area, epidermis, and central cylinder of the PRs, and LN significantly up-regulated the expression of ZmPIN1a in PR tip and LRP initiation zone, (Fig. 5). Considering the increased ZmPIN1a expression increased auxin accumulated in root tips , and LN treatment enhanced IAA levels in PR tips (Fig. 2). We assumed that ZmPIN1a modulates auxin reallocation involved in nitrate-mediated root formation. Here, ZmPIN1a-OE plants had higher IAA efflux in root tip and influx in LRP initiation zone under LN and NN conditions (Fig. 7A-B). Correspondingly, overexpression of ZmPIN1a enhanced IAA accumulation in root tip, while decreased IAA levels in the LRP initiation zone (Fig. 7C-D). These findings suggested that LN treatment affected the expression of ZmPIN1a gene, the expression of ZmPIN1a gene promoted the net auxin flux and auxin transport, further leaded to an evident enhancement of IAA levels in root tips.
Furthermore, overexpression of ZmPIN1a enhanced PR elongation and repressed LR formation, and the PR elongation and LR formation in ZmPIN1a-OE plants exhibited a higher degree of sensitivity under LN condition (Fig. 6). More importantly, the inhibition of PR length of ZmPIN1a-OE plants was significantly higher than that of wild-type by NPA, and the inhibition rate was significantly higher under LN condition than that under NN condition, and similar observations were presented in LRP density and number of ZmPIN1a-OE plants (Fig. 8A-C). These indicated that ZmPIN1a was essential to modulate auxin levels for building the root architecture in response to N availability. The interpretation of these observations was that ZmPIN1a acted to drain auxin from the LRP initiation zone to root tips, resulting in the auxin level accumulation in root tips under LN condition. However, in the LRP initiation zone, auxin falling below a threshold sufficient to support normal LRP initiation. This result provided further evidence about the role of auxin polar transport in NO3–-mediated root growth in maize. How ZmPIN1a regulated auxin transport from LRP initiation to root tips in the process of LN-mediated root development needs to be explored in future.
Taken together, our results suggested that the auxin efflux carriers ZmPIN1a played an important role in LN-mediated PR elongation coupled with LR inhibition via altering the auxin levels and transport in root tip and LRP initiation zone. Our studies provide valuable knowledge that would pave the way for expediting crop breeding programs involving genetic improvement in N use efficiency through root architecture modifications.
Materials and methods
Plant materials and growth conditions
ZmPIN1a-overexpressing plants (OE 1, OE 3, and OE 5) were obtained from Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University. The lines were produced in maize inbred line B73-329 background. For hydroponics, seeds of the maize varieties ZD958, B73-329 and transgenic lines were surface sterilized in 10% (v/v) H2O2 for 20 min, rinsed with distilled water five times and then sown in the sand. Seven days after germination, the endosperm of each seeding was excised, and uniform seedings with two visible leaves were transferred to a hydroponic box containing 5 L nutrient solution. The experiment was conducted in a growth chamber with 16-h-light (28 °C)/8-h-dark (22 °C) and the relative humidity controlled to approximately 70 to 80%. The basic nutrient solution contained 2 mM KNO3, 1 mM CaCl2, 0.1 mM KH2PO4, 0.5 mM MgSO4, 0.1 mM EDTA-Fe, 0.03 mM H3BO3, 0.0025 mM ZnSO4, 0.008 mM CuSO4, 0.005 mM MnSO4 and 0.0003 mM (NH4)6Mo7O24. Plants were supplied with half-strength nutrient solution for 2 d and then transferred into a solution with either low NO3– (LN, 0.05 mM) or normal NO3– (NN, 2 mM) as control. To exclude the possibility of potassium (K+) interference, the concentrations of K+ in the LN solution were supplemented to the same levels as those of the NN solution using KCl. For all growth solutions, the pH was adjusted to 5.8–6.0 with KOH. The nutrient solution was renewed every other day and aerated continuously by an electric pump.
Measurement of root morphology
The seedlings were sampled after N treatment. The PR length was measured with a ruler. The total root length was measured with the scanner (Epson V700, Beijing, China) and the images were analysed using WinRHIZO Pro 5.0 (Quebec City, Canada). Feulgen staining was used to estimate the number and density of lateral root primordium (LRP) in the apical unbranched zone of the PR.
Measurement of IAA flux in roots
Indole-3-acetic acid (IAA) flux in roots was measured using Non-invasive Micro-test Technology (NMT) system (Younger USA LLC, Amherst, MA 01,002, USA; youngeru-sa.com) by Xuyue (Beijing) Sci. & Tech. Co., Ltd., Beijing, China, as described previously [35, 36]. For each experimental group, 3 uniform seedlings were selected at 2 d after LN treatment, and the roots were immediately equilibrated for 15 min in the test buffer (0.05/2 mM KNO3, 0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.3 mM MES, pH 6.0). The IAA fluxes were determined at concentrating on the following zone including 100, 200, 400, 700, 1500 and 2000 μm from the root tip, and then the IAA flux was measured at 400 and 1500 μm from the root tip. IAA fluxes were recorded continuously for 3 min to yield a steady mean value. At least three individual plants were measured for each treatment. The positive and negative values indicated net IAA efflux and influx, respectively.
In order to observe the auxin signaling response to LN treatment in the roots, the transgenic line carrying DR5rev::RFP auxin response marker with a synthetic auxin promoter  was used. Following the exposure of DR5rev::RFP seedlings to LN treatment for 24 h, the root tip were subsequently scanned for the RFP signal using Zeiss LSM 880.
Determination of IAA concentration
Two segments were collected from the roots at 2 d after LN treatment. The first segment (0–5 mm root tip) was as the root meristem controlling root growth, and other segment (5–25 mm behind the root tip of the PR) was the LRP initiation and development zone. The free IAA was quantified using high-performance liquid chromatog-raphy-electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS). The root samples were ground to fine powder in liquid nitrogen and each sample (50 mg) was transferred to 1.5 mL tubes and added 50 µL of the working solution of internal standards (2H-labeled IAA). Subsequently, 500 µL of extraction solvent (2-propanol/H2O/concentrated HCl, 2:1:0.002, v/v/v) was added to each sample and vortexed. The remaining steps were performed as previously reported by .
RNA isolation and quantitative RT-PCR (qRT-PCR) analysis
Total RNA was extracted from the roots using the Plant RNeasy Mini kit (Tiangen). For RT-qPCR analysis, 1ug total RNA was reverse transcribed into cDNA using Oligo d (T) 18 primer and M-MLV reverse transcriptase (Takara, Japan). The diluted cDNA was amplified using SYBR Premix EX TaqTM (Takara, Japan) on an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, USA) following the manufacturer’s protocol. ZmUBC (ubiquitin C) was used to normalize gene expression. Fold change of gene expression values were calculated using the 2−ΔΔCT methods . All primers were listed in Table S2.
In-situ hybrid dization
In-situ hybridization experiments have been described previously  with some modifications. The PR tip were harvested at 12 h after LN treatment, fixed in 3.7% for-mal-acetic-alcohol, vacuum filtered for 15 min, and fixed overnight in a fresh fixative solution. The samples were dehydrated with a graded series of ethanol and xylene, and finally embedded in paraffin. The 10 µm-thick root sections obtained using Microtome (Leica Germany) were transferred to ice-cold DEPC water. Digoxigenin-labeled probes were synthesized for cDNA using gene-specific primers, including SP6 and T7 RNA polymerase binding sites. The sense and antisense probes were generated using DIG RNA Labeling Kit (SP6/T7, Roche). The sense probe was produced with SP6 RNA polymerase as negative control. Other steps were performed as previously reported . The details of the primers used for probe synthesis are given in Table S2.
Nutrient solution treatments with NAA and NPA
Maize ZD958 seedlings were treated with naphthylacetic acid (NAA; an analogue of IAA) and auxin-transport inhibitor NPA when the nutrient solution was changed every time. The final concentration of NAA was 1 μM, NPA was 0.1 μM. NAA and NPA were first dissolved in dimethyl sulfoxide (DMSO). The same dose of DMSO was added to the control.
Lanolin paste treatment of NPA
For the experiment of ZmPIN1a-OE plants applied with NPA treatment, ZmPIN1a-OE plants were grown in half-strength nutrient solution for 2 d, the lanolin paste containing NPA was smeared uniformly surround the hypocotyl of plants when the nutrient solution was changed every time. The concentration of NPA was 1000 μM. The control plants were treated with lanolin paste without NPA. The method was performed as previously reported by .
The data were statistically analysed using the SAS 9.0 (SAS Institute Ltd., USA). The student’s test was used for the comparisons between two groups of date. For the data sets of more than two groups, one-way ANOVA with LSD (P ≤ 0.05) was used.
Availability of data and materials
All data generated or analyzed during this study are available from the corresponding author on reasonable request.
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We appreciate Dr. Zhangkui Wang at China Agricultural University for providing maize seeds of the DR5rev: RFP reporter which were generated in Dr Gallavotti Andrea’s Lab. Seeds of B73-329, ZmPIN1a-overexpressing plants were produced by Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.
This work was supported by Natural Science Foundation of Shandong Province, China (Grant No. ZR2021QC090) and National Nature Science Foundation of China (Grant No. 31871546).
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Additional file 1:
Fig. S1. Effects of auxin on root growth under LN and NN conditions. Fig. S2. Effects of auxin inhibitor NPA on root growth under LN and NN conditions. Fig. S3. Relative expression level of ZmPIN1a in roots of ZmPIN1a-OE plants. Table S1. Effects of LN supply on total root length. Table S2. List of primers used in this study.
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Wang, Y., Xing, J., Wan, J. et al. Auxin efflux carrier ZmPIN1a modulates auxin reallocation involved in nitrate-mediated root formation. BMC Plant Biol 23, 74 (2023). https://doi.org/10.1186/s12870-023-04087-0
- Auxin efflux carrier ZmPIN1a
- Low nitrate
- Root formation