Excessive iron deposition in root apoplast is involved in growth arrest of roots in response to low pH.

The rhizotoxicity of protons (H+) in acidic soils is a fundamental constraint that results in serious yield losses. However, the mechanisms underlying H+-mediated inhibition of root growth are poorly understood. In this study, we revealed that H+-induced root growth inhibition in Arabidopsis depends considerably on excessive iron deposition in the root apoplast. Reducing such aberrant iron deposition by decreasing the iron supply or disrupting the ferroxidases LOW PHOSPHATE ROOT 1 (LPR) and LPR2 attenuates the inhibitory effect of H+ on primary root growth efficiently. Further analysis showed that excessive iron deposition triggers a burst of highly reactive oxygen species, consequently impairing normal root development. Our study uncovered a valuable strategy for improving the ability of plants to tolerate H+ toxicity by manipulating iron availability.

STOP1 activates NRT1.1-mediated nitrate uptake to create a favorable rhizospheric pH for plant adaptation to acidity.

Protons (H+) in acidic soils arrest plant growth. However, the mechanisms by which plants optimize their biological processes to diminish the unfavorable effects of H+ stress remain largely unclear. Here, we showed that in the roots of Arabidopsis thaliana, the C2H2-type transcription factor STOP1 in the nucleus was enriched by low pH in a nitrate-independent manner, with the spatial expression pattern of NITRATE TRANSPORTER 1.1 (NRT1.1) established by low pH required the action of STOP1. Additionally, the nrt1.1 and stop1 mutants, as well as the nrt1.1 stop1 double mutant, had a similar hypersensitive phenotype to low pH, indicating that STOP1 and NRT1.1 function in the same pathway for H+ tolerance. Molecular assays revealed that STOP1 directly bound to the promoter of NRT1.1 to activate its transcription in response to low pH, thus upregulating its nitrate uptake. This action improved the nitrogen use efficiency (NUE) of plants and created a favorable rhizospheric pH for root growth by enhancing H+ depletion in the rhizosphere. Consequently, the constitutive expression of NRT1.1 in stop1 mutants abolished the hypersensitive phenotype to low pH. These results demonstrate that STOP1-NRT1.1 is a key module for plants to optimize NUE and ensure better plant growth in acidic media.

Inhibition of shoot-expressed NRT1.1 improves reutilization of apoplastic iron under iron-deficient conditions.

Iron deficiency is a major constraint for plant growth in calcareous soils. The interplay between NO3 - and Fe nutrition affects plant performance under Fe-deficient conditions. However, how NO3 - negatively regulates Fe nutrition at the molecular level in plants remains elusive. Here, we showed that the key nitrate transporter NRT1.1 in Arabidopsis plants, especially in the shoots, was markedly downregulated at post-translational levels by Fe deficiency. However, loss of NRT1.1 function alleviated Fe deficiency chlorosis, suggesting that downregulation of NRT1.1 by Fe deficiency favors plant tolerance to Fe deficiency. Further analysis showed that although disruption of NRT1.1 did not alter Fe levels in both the shoots and roots, it improved the reutilization of apoplastic Fe in shoots but not in roots. In addition, disruption of NRT1.1 prevented Fe deficiency-induced apoplastic alkalization in shoots by inhibiting apoplastic H+ depletion via NO3 - uptake. In vitro analysis showed that reduced pH facilitates release of cell wall-bound Fe. Thus, foliar spray with an acidic buffer promoted the reutilization of Fe in the leaf apoplast to enhance plant tolerance to Fe deficiency, while the opposite was true for the foliar spray with a neutral buffer. Thus, downregulation of the shoot-part function of NRT1.1 prevents apoplastic alkalization to ensure the reutilization of apoplastic Fe under Fe-deficient conditions. Our findings may provide a basis for elucidating the link between N and Fe nutrition in plants and insight to scrutinize the relevance of shoot-expressed NRT1.1 to the plant response to stress.

The K+ and NO3 - Interaction Mediated by NITRATE TRANSPORTER1.1 Ensures Better Plant Growth under K+-Limiting Conditions.

K+ and NO3 - are the major forms of potassium and nitrogen that are absorbed by the roots of most terrestrial plants. In this study, we observed that a close relationship between NO3 - and K+ in Arabidopsis (Arabidopsis thaliana) is mediated by NITRATE TRANSPORTER1.1 (NRT1.1). The nrt1.1 knockout mutants showed disturbed K+ uptake and root-to-shoot allocation, and were characterized by growth arrest under K+-limiting conditions. The K+ uptake and root-to-shoot allocation of these mutants were partially recovered by expressing NRT1.1 in the root epidermis-cortex and central vasculature using SULFATE TRANSPORTER1;2 and PHOSPHATE1 promoters, respectively. Two-way analysis of variance based on the K+ contents in nrt1.1-1/K + transporter1, nrt1.1-1/high-affinity K + transporter5-3, nrt1.1-1/K + uptake permease7, and nrt1.1-1/stelar K + outward rectifier-2 double mutants and the corresponding single mutants and wild-type plants revealed physiological interactions between NRT1.1 and K+ channels/transporters located in the root epidermis-cortex and central vasculature. Further study revealed that these K+ uptake-related interactions are dependent on an H+-consuming mechanism associated with the H+/NO3 - symport mediated by NRT1.1. Collectively, these data indicate that patterns of NRT1.1 expression in the root epidermis-cortex and central vasculature are coordinated with K+ channels/transporters to improve K+ uptake and root-to-shoot allocation, respectively, which in turn ensures better growth under K+-limiting conditions.

Inhibition of DNA demethylation enhances plant tolerance to cadmium toxicity by improving iron nutrition.

Although the alteration of DNA methylation due to abiotic stresses, such as exposure to the toxic metal cadmium (Cd), has been often observed in plants, little is known about whether such epigenetic changes are linked to the ability of plants to adapt to stress. Herein, we report a close linkage between DNA methylation and the adaptational responses in Arabidopsis plants under Cd stress. Exposure to Cd significantly inhibited the expression of three DNA demethylase genes ROS1/DML2/DML3 (RDD) and elevated DNA methylation at the genome-wide level in Col-0 roots. Furthermore, the profile of DNA methylation in Cd-exposed Col-0 roots was similar to that in the roots of rdd triple mutants, which lack RDD, indicating that Cd-induced DNA methylation is associated with the inhibition of RDD. Interestingly, the elevation in DNA methylation in rdd conferred a higher tolerance against Cd stress and improved cellular Fe nutrition in the root tissues. In addition, lowering the Fe supply abolished improved Cd tolerance due to the lack of RDD in rdd. Together, these data suggest that the inhibition of RDD-mediated DNA demethylation in the roots by Cd would in turn enhance plant tolerance to Cd stress by improving Fe nutrition through a feedback mechanism.

Nitrate transporter 1.1 alleviates lead toxicity in Arabidopsis by preventing rhizosphere acidification.

Identification of the mechanisms that control lead (Pb) concentration in plants is a prerequisite for minimizing dietary uptake of Pb from contaminated crops. This study examines how nitrate uptake by roots affects Pb uptake and reveals a new resistance strategy for plants to cope with Pb contamination. We investigated the interaction between nitrate transporter (NRT)-mediated NO3- uptake and exposure to Pb in Arabidopsis using NRT-related mutants. Exposure to Pb specifically stimulated NRT1.1-mediated nitrate uptake. Loss of function of NRT1.1 in nrt1.1-knockout mutants resulted in greater Pb toxicity and higher Pb accumulation in nitrate-sufficient growth medium, whereas no difference was seen between wild-type plants and null-mutants for NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5. These results indicate that only NRT1.1-mediated NO3- uptake alleviated Pb toxicity in the plants. Further examination indicated that rhizosphere acidification, which favors Pb entry to roots by increasing its availability, is prevented when NRT1.1 is functional and both NO3- and NH4+ are present in the medium.

Ethylene and nitric oxide interact to regulate the magnesium deficiency-induced root hair development in Arabidopsis.

Nitric oxide (NO) and ethylene respond to biotic and abiotic stresses through either similar or independent processes. This study examines the mechanism underlying the effects of NO and ethylene on promoting root hair development in Arabidopsis under magnesium (Mg) deficiency. The interaction between NO and ethylene in the regulation of Mg deficiency-induced root hair development was investigated using NO- and ethylene-related mutants and pharmacological methods. Mg deficiency triggered a burst of NO and ethylene, accompanied by a stimulated development of root hairs. Interestingly, ethylene facilitated NO generation by activation of both nitrate reductase and nitric oxide synthase-like (NOS-L) in the roots of Mg-deficient plants. In turn, NO enhanced ethylene synthesis through stimulating the activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase and ACC synthase (ACS). These two processes constituted an NO-ethylene feedback loop. Blocking either of these two processes inhibited the stimulation of root hair development under Mg deficiency. In conclusion, we suggest that Mg deficiency increases the production of NO and ethylene in roots, each influencing the accumulation and role of the other, and thus these two signals interactively regulate Mg deficiency-induced root hair morphogenesis.

Increased Sucrose Accumulation Regulates Iron-Deficiency Responses by Promoting Auxin Signaling in Arabidopsis Plants.

Previous studies have identified that auxins acts upstream of nitric oxide in regulating iron deficiency responses in roots, but the upstream signaling molecule of auxins remains unknown. In this study, we showed that Fe deficiency increased sucrose (Suc) level in roots of Arabidopsis (Arabidopsis thaliana). Exogenous application of Suc further stimulated Fe deficiency-induced ferric-chelate-reductase (FCR) activity and expression of Fe acquisition-related genes FRO2, IRT1, and FIT in roots. The opposite patterns were observed in the dark treatment. In addition, FCR activity and expression of Fe acquisition-related genes were higher in the Suc high-accumulating transgenic plant 35S::SUC2 but were lower in the Suc low-accumulating mutant suc2-5 compared with wild-type plants under Fe-deficient conditions. Consequently, Fe deficiency tolerance was enhanced in 35S::SUC2 but was compromised in suc2-5. Exogenous Suc also increased root ß-glucuronidase (GUS) activity in auxin-inducible reporter DR5-GUS transgenic plants under Fe deficiency. However, exogenous Suc failed to increase FCR activity and expression of Fe acquisition-related genes in the auxin transport-impaired mutants aux1-7 and pin1-1 as well as in the wild-type plants treated with an auxin transport inhibitor under Fe deficiency. In summary, we found that increased Suc accumulation is required for regulating Fe deficiency responses in plants, with auxins acting downstream in transmitting the Fe deficiency signal.

Alleviation of proton toxicity by nitrate uptake specifically depends on nitrate transporter 1.1 in Arabidopsis.

Protons in acid soil are highly rhizotoxic to plants, but the mechanism of tolerance of plants to protons is largely unknown. Nitrate uptake by root cells is accompanied by the uptake of protons. Therefore, nitrate uptake transporters (NRTs) may be involved in plant tolerance to proton toxicity. We investigated the root nitrate uptake response to proton stress in Arabidopsis and its association with proton tolerance using NRT-related mutants and pharmacological methods. Lack of NRT1.1 in knockout nrt1.1 mutants led to impaired proton tolerance in nitrate-sufficient growth medium, whereas no difference was seen between wild-type plants and NRT1.2-, NRT2.1-, NRT2.2-, and NRT2.4-null mutants. Another nrt1.1 point mutant, which is defective in nitrate uptake but has a normal nitrate-sensing function, also had impaired proton tolerance compared with the wild-type plant. Furthermore, proton stress induced NRT1.1-mediated nitrate uptake. These results indicate that NRT1.1-conferred proton tolerance depends on nitrate uptake activity. In addition, the rooting medium was alkalified by wild-type plants, but not by knockout nrt1.1 mutants, and in pH-buffered medium, there were no differences in proton tolerance between wild-type plants and knockout nrt1.1 mutants. We conclude that NRT1.1-mediated nitrate uptake plays a crucial role in plant proton tolerance by alkalifying the rhizosphere.

Inhibition of nitrate transporter 1.1-controlled nitrate uptake reduces cadmium uptake in Arabidopsis.

Identification of mechanisms that decrease cadmium accumulation in plants is a prerequisite for minimizing dietary uptake of cadmium from contaminated crops. Here, we show that cadmium inhibits nitrate transporter 1.1 (NRT1.1)-mediated nitrate (NO3 (-)) uptake in Arabidopsis (Arabidopsis thaliana) and impairs NO3 (-) homeostasis in roots. In NO3 (-)-containing medium, loss of NRT1.1 function in nrt1.1 mutants leads to decreased levels of cadmium and several other metals in both roots and shoots and results in better biomass production in the presence of cadmium, whereas in NO3 (-)-free medium, no difference is seen between nrt1.1 mutants and wild-type plants. These results suggest that inhibition of NRT1.1 activity reduces cadmium uptake, thus enhancing cadmium tolerance in an NO3 (-) uptake-dependent manner. Furthermore, using a treatment rotation system allowing synchronous uptake of NO3 (-) and nutrient cations and asynchronous uptake of cadmium, the nrt1.1 mutants had similar cadmium levels to wild-type plants but lower levels of nutrient metals, whereas the opposite effect was seen using treatment rotation allowing synchronous uptake of NO3 (-) and cadmium and asynchronous uptake of nutrient cations. We conclude that, although inhibition of NRT1.1-mediated NO3 (-) uptake by cadmium might have negative effects on nitrogen nutrition in plants, it has a positive effect on cadmium detoxification by reducing cadmium entry into roots. NRT1.1 may regulate the uptake of cadmium and other cations by a common mechanism.

Oxalate synthesis in leaves is associated with root uptake of nitrate and its assimilation in spinach (Spinacia oleracea L.) plants.

BACKGROUND: Excessive accumulation of oxalate in numerous vegetables adversely affects their quality as food. While it is known that nitrate could effectively stimulate oxalate accumulation in many vegetables, little information is available about the mechanism of nitrate-induced oxalate accumulation. In this study, we examined the association of oxalate synthesis with nitrate uptake and assimilation in two genotypes of spinach (Spinacia oleracea L.), Heizhenzhu and Weilv. RESULTS: Increasing nitrate levels resulted in enhanced synthesis of oxalate, as well as increased root uptake of nitrate and leaf activities of nitrate reductase (NR) and glutamine synthetase (GS) for both genotypes. Correlation analysis revealed that oxalate accumulation in spinach leaves was positively related with rate of nitrate uptake by roots, as well as leaf activities of NR and GS. Addition of plasmalemma H(+)-ATPase inhibitor sodium vanadate (Na3VO4) significantly decreased leaf oxalate accumulation in both genotypes. Presence of NR or GS inhibitors led to reduction of leaf oxalate contents, GS/NR activities and decreased nitrate uptake rate. Significantly higher levels of nitrate root uptake, leaf NR and GS activities were observed in the high-oxalate genotype Heizhenzhu than in Weilv. CONCLUSION: Oxalate synthesis in leaves of spinach is not only positively associated with root uptake of nitrate, but also with its assimilation within the plants.

An underground tale: contribution of microbial activity to plant iron acquisition via ecological processes.

BACKGROUND: Iron (Fe) deficiency in crops is a worldwide agricultural problem. Plants have evolved several strategies to enhance Fe acquisition, but increasing evidence has shown that the intrinsic plant-based strategies alone are insufficient to avoid Fe deficiency in Fe-limited soils. Soil micro-organisms also play a critical role in plant Fe acquisition; however, the mechanisms behind their promotion of Fe acquisition remain largely unknown. SCOPE: This review focuses on the possible mechanisms underlying the promotion of plant Fe acquisition by soil micro-organisms. CONCLUSIONS: Fe-deficiency-induced root exudates alter the microbial community in the rhizosphere by modifying the physicochemical properties of soil, and/or by their antimicrobial and/or growth-promoting effects. The altered microbial community may in turn benefit plant Fe acquisition via production of siderophores and protons, both of which improve Fe bioavailability in soil, and via hormone generation that triggers the enhancement of Fe uptake capacity in plants. In addition, symbiotic interactions between micro-organisms and host plants could also enhance plant Fe acquisition, possibly including: rhizobium nodulation enhancing plant Fe uptake capacity and mycorrhizal fungal infection enhancing root length and the nutrient acquisition area of the root system, as well as increasing the production of Fe(3+) chelators and protons.

Short-term alteration of nitrogen supply prior to harvest affects quality in hydroponic-cultivated spinach (Spinacia oleracea).

BACKGROUND: Quality-associated problems, such as excessive in planta accumulation of oxalate, often arise in soillessly cultivated spinach (Spinacia oleracea). Maintaining a higher level of ammonium (NH4⁺) compared to nitrate (NO3⁻) during the growth period can effectively decrease the oxalate content in hydroponically cultivated vegetables. However, long-term exposure to high concentrations of NH4⁺ induces toxicity in plants, and thus decreases the biomass production. Short-term application of NH4⁺ before harvesting in soilless cultivation may provide an alternative strategy to decrease oxalate accumulation in spinach, and minimise the yield reduction caused by NH4⁺ toxicity. RESULT: The plants were pre-cultured in 8 mmol L⁻¹ NO3⁻ nutrient solution. Next, 6 days before harvest, the plants were transferred to a nutrient solution containing 4 mmol L⁻¹ NO3⁻ and 4 mmol L⁻¹ NH4⁺. This new mix clearly reduced oxalate accumulation, increased levels of several antioxidant compounds, and enhanced antioxidant capacity in the edible parts of spinach plants, but it did not affect biomass production. However, when the 8 mmol L⁻¹ NO3⁻ was shifted to either nitrogen-free, 4 mmol L⁻¹ NH4⁺ or 8 mmol L⁻¹ NH4⁺ treatments, although some of the quality indexes were improved, yields were significantly reduced. CONCLUSIONS: Short-term alteration of nitrogen supply prior to harvest significantly affects quality and biomass of spinach plants, and we strongly recommend to simultaneously use NO3⁻ and NH4⁺ in hydroponic cultivation, which improves vegetable quality without decreasing biomass production.

The 14-3-3 protein GENERAL REGULATORY FACTOR11 (GRF11) acts downstream of nitric oxide to regulate iron acquisition in Arabidopsis thaliana.

Here we report the function of a general regulatory factor, GENERAL REGULATORY FACTOR11 (GRF11), in terms of the iron (Fe) deficiency response. Physiological and molecular responses of the loss-of-function Arabidopsis thaliana grf11 mutant to Fe supply were investigated. Genes involved in posttranscriptional regulation of FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) were also analyzed. In addition, the molecular link between the signaling molecule nitric oxide (NO) and Fe deficiency responses was further dissected. Our results suggest that GRF11 is necessary for induction of Fe-deficiency-tolerance mechanisms. The FIT protein can bind to the promoter of GRF11, which contains an E-box motif. GRF11 also positively affects FIT transcription but has no influence on the genes involved in posttranscriptional regulation of FIT. Furthermore, NO positively regulates GRF11 induction upon the onset of Fe deficiency. We propose that, upon the onset of Fe deficiency, induction of FIT expression is dependent on GRF11, which acts downstream of NO to mediate Fe deficiency responses.

Beyond iron-storage pool: functions of plant apoplastic iron during stress.

Iron (Fe) is an essential micronutrient for plants, and its storage in the apoplast represents an important Fe pool. Plants have developed various strategies to reutilize this apoplastic Fe pool to adapt to Fe deficiency. In addition, growing evidence indicates that the dynamic changes in apoplastic Fe are critical for plant adaptation to other stresses, including ammonium stress, phosphate deficiency, and pathogen attack. In this review, we discuss and scrutinize the relevance of apoplastic Fe for plant behavior changes in response to stress cues. We mainly focus on the relevant components that modulate the actions and downstream events of apoplastic Fe in stress signaling networks.

Iron uptake system mediates nitrate-facilitated cadmium accumulation in tomato (Solanum lycopersicum) plants.

Nitrogen (N) management is a promising agronomic strategy to minimize cadmium (Cd) contamination in crops. However, it is unclear how N affects Cd uptake by plants. Wild-type and iron uptake-inefficient tomato (Solanum lycopersicum) mutant (T3238fer) plants were grown in pH-buffered hydroponic culture to investigate the direct effect of N-form on Cd uptake. Wild-type plants fed NO3⁻ accumulated more Cd than plants fed NH4⁺. Iron uptake and LeIRT1 expression in roots were also greater in plants fed NO3⁻. However, in mutant T3238fer which loses FER function, LeIRT1 expression in roots was almost completely terminated, and the difference between NO3⁻ and NH4⁺ treatments vanished. As a result, the N-form had no effect on Cd uptake in this mutant. Furthermore, suppression of LeIRT1 expression by NO synthesis inhibition with either tungstate or L-NAME, also substantially inhibited Cd uptake in roots, and the difference between N-form treatments was diminished. Considering all of these findings, it was concluded that the up-regulation of the Fe uptake system was responsible for NO3⁻-facilitated Cd accumulation in plants.

Nitrogen in plants: from nutrition to the modulation of abiotic stress adaptation.

Nitrogen is one of the most important nutrient for plant growth and development; it is strongly associated with a variety of abiotic stress responses. As sessile organisms, plants have evolved to develop efficient strategies to manage N to support growth when exposed to a diverse range of stressors. This review summarizes the recent progress in the field of plant nitrate (NO3-) and ammonium (NH4+) uptake, which are the two major forms of N that are absorbed by plants. We explore the intricate relationship between NO3-/NH4+ and abiotic stress responses in plants, focusing on stresses from nutrient deficiencies, unfavorable pH, ions, and drought. Although many molecular details remain unclear, research has revealed a number of core signaling regulators that are associated with N-mediated abiotic stress responses. An in-depth understanding and exploration of the molecular processes that underpin the interactions between N and abiotic stresses is useful in the design of effective strategies to improve crop growth, development, and productivity.

Phloem iron remodels root development in response to ammonium as the major nitrogen source.

Plants use nitrate and ammonium as major nitrogen (N) sources, each affecting root development through different mechanisms. However, the exact signaling pathways involved in root development are poorly understood. Here, we show that, in Arabidopsis thaliana, either disruption of the cell wall-localized ferroxidase LPR2 or a decrease in iron supplementation efficiently alleviates the growth inhibition of primary roots in response to NH4+ as the N source. Further study revealed that, compared with nitrate, ammonium led to excess iron accumulation in the apoplast of phloem in an LPR2-dependent manner. Such an aberrant iron accumulation subsequently causes massive callose deposition in the phloem from a resulting burst of reactive oxygen species, which impairs the function of the phloem. Therefore, ammonium attenuates primary root development by insufficiently allocating sucrose to the growth zone. Our results link phloem iron to root morphology in response to environmental cues.

The ferroxidases are critical for Fe(II) oxidation in xylem to ensure a healthy Fe allocation in Arabidopsis thaliana.

The long-distance transport of iron (Fe) in the xylem is critical for maintaining systemic Fe homeostasis in plants. The loading form of Fe(II) into the xylem and the long-distance translocation form of Fe(III)-citrate have been identified, but how Fe(II) is oxidized to Fe(III) in the xylem remains unknown. Here, we showed that the cell wall-resided ferroxidases LPR1 and LPR2 (LPRs) were both specifically expressed in the vascular tissues of Arabidopsis thaliana, while disruption of both of them increased Fe(II) in the xylem sap and caused excessive Fe deposition in the xylem vessel wall under Fe-sufficient conditions. As a result, a large amount of Fe accumulated in both roots and shoots, hindering plant growth. Moreover, under low-Fe conditions, LPRs were preferentially induced in old leaves, but the loss of LPRs increased Fe deposition in the vasculature of older leaves and impeded Fe allocation to younger leaves. Therefore, disruption of both LPRs resulted in severer chlorosis in young leaves under Fe-deficient conditions. Taken together, the oxidation of Fe(II) to Fe(III) by LPRs in the cell wall of vasculature plays an important role in xylem Fe allocation, ensuring healthy Fe homeostasis for normal plant growth.

Nitric oxide acts downstream of auxin to trigger root ferric-chelate reductase activity in response to iron deficiency in Arabidopsis.

In response to iron (Fe) deficiency, dicots employ a reduction-based mechanism by inducing ferric-chelate reductase (FCR) at the root plasma membrane to enhance Fe uptake. However, the signal pathway leading to FCR induction is still unclear. Here, we found that the Fe-deficiency-induced increase of auxin and nitric oxide (NO) levels in wild-type Arabidopsis (Arabidopsis thaliana) was accompanied by up-regulation of root FCR activity and the expression of the basic helix-loop-helix transcription factor (FIT) and the ferric reduction oxidase 2 (FRO2) genes. This was further stimulated by application of exogenous auxin (α-naphthaleneacetic acid) or NO donor (S-nitrosoglutathione [GSNO]), but suppressed by either polar auxin transport inhibition with 1-naphthylphthalamic acid or NO scavenging with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, tungstate, or N(ω)-nitro-L-arginine methyl ester hydrochloride. On the other hand, the root FCR activity, NO level, and gene expression of FIT and FRO2 were higher in auxin-overproducing mutant yucca under Fe deficiency, which were sharply restrained by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide treatment. The opposite response was observed in a basipetal auxin transport impaired mutant aux1-7, which was slightly rescued by exogenous GSNO application. Furthermore, Fe deficiency or α-naphthaleneacetic acid application failed to induce Fe-deficiency responses in noa1 and nial nia2, two mutants with reduced NO synthesis, but root FCR activities in both mutants could be significantly elevated by GSNO. The inability to induce NO burst and FCR activity was further verified in a double mutant yucca noa1 with elevated auxin production and reduced NO accumulation. Therefore, we presented a novel signaling pathway where NO acts downstream of auxin to activate root FCR activity under Fe deficiency in Arabidopsis.