OsMADS5 interacts with OsSPL14/17 to inhibit rice root elongation by restricting cell proliferation of root meristem under ammonium supply.

Nitrogen (N) is a vital major nutrient for rice (Oryza sativa). Rice responds to different applications of N by altering its root morphology, including root elongation. Although ammonium ( NH 4 + ) is the primary source of N for rice, NH 4 + is toxic to rice roots and inhibits root elongation. However, the precise molecular mechanism that NH 4 + -inhibited root elongation of rice is not well understood. Here, we identified a rice T-DNA insert mutant of OsMADS5 with a longer seminal root (SR) under sufficient N conditions. Reverse-transcription quantitative PCR analysis revealed that the expression level of OsMADS5 was increased under NH 4 + compared with NO 3 - supply. Under NH 4 + conditions, knocking out OsMADS5 (cas9) produced a longer SR, phenocopying osmads5, while there was no significant difference in SR length between wild-type and cas9 under NO 3 - supply. Moreover, OsMADS5-overexpression plants displayed the opposite SR phenotype. Further study demonstrated that enhancement of OsMADS5 by NH 4 + supply inhibited rice SR elongation, likely by reducing root meristem activity of root tip, with the involvement of OsCYCB1;1. We also found that OsMADS5 interacted with OsSPL14 and OsSPL17 (OsSPL14/17) to repress their transcriptional activation by attenuating DNA binding ability. Moreover, loss of OsSPL14/17 function in osmads5 eliminated its stimulative effect on SR elongation under NH 4 + conditions, implying OsSPL14/17 may function downstream of OsMADS5 to mediate rice SR elongation under NH 4 + supply. Overall, our results indicate the existence of a novel modulatory pathway in which enhancement of OsMADS5 by NH 4 + supply represses the transcriptional activities of OsSPL14/17 to restrict SR elongation of rice.

The D14-SDEL1-SPX4 cascade integrates the strigolactone and phosphate signalling networks in rice.

Modern agriculture needs large quantities of phosphate (Pi) fertilisers to obtain high yields. Information on how plants sense and adapt to Pi is required to enhance phosphorus-use efficiency (PUE) and thereby promote agricultural sustainability. Here, we show that strigolactones (SLs) regulate rice root developmental and metabolic adaptations to low Pi, by promoting efficient Pi uptake and translocation from roots to shoots. Low Pi stress triggers the synthesis of SLs, which dissociate the Pi central signalling module of SPX domain-containing protein (SPX4) and PHOSPHATE STARVATION RESPONSE protein (PHR2), leading to the release of PHR2 into the nucleus and activating the expression of Pi-starvation-induced genes including Pi transporters. The SL synthetic analogue GR24 enhances the interaction between the SL receptor DWARF 14 (D14) and a RING-finger ubiquitin E3 ligase (SDEL1). The sdel mutants have a reduced response to Pi starvation relative to wild-type plants, leading to insensitive root adaptation to Pi. Also, SLs induce the degradation of SPX4 via forming the D14-SDEL1-SPX4 complex. Our findings reveal a novel mechanism underlying crosstalk between the SL and Pi signalling networks in response to Pi fluctuations, which will enable breeding of high-PUE crop plants.

The miR167-OsARF12 module regulates rice grain filling and grain size downstream of miR159.

Grain weight and quality are always determined by grain filling. Plant microRNAs have drawn attention as key targets for regulation of grain size and yield. However, the mechanisms that underlie grain size regulation remain largely unclear because of the complex networks that control this trait. Our earlier studies demonstrated that suppressed expression of miR167 (STTM/MIM167) substantially increased grain weight. In a field test, the yield increased up to 12.90%-21.94% because of a significantly enhanced grain filling rate. Here, biochemical and genetic analyses revealed the regulatory effects of miR159 on miR167 expression. Further analysis indicated that OsARF12 is the major mediator by which miR167 regulates rice grain filling. Overexpression of OsARF12 produced grain weight and grain filling phenotypes resembling those of STTM/MIM167 plants. Upon in-depth analysis, we found that OsARF12 activates OsCDKF;2 expression by directly binding to the TGTCGG motif in its promoter region. Flow cytometry analysis of young panicles from OsARF12-overexpressing plants and examination of cell number in cdkf;2 mutants verified that OsARF12 positively regulates grain filling and grain size by targeting OsCDKF;2. Moreover, RNA sequencing results suggested that the miR167-OsARF12 module is involved in the cell development process and hormone pathways. OsARF12-overexpressing plants and cdkf;2 mutants exhibited enhanced and reduced sensitivity to exogenous auxin and brassinosteroid (BR) treatment, confirming that targeting of OsCDKF;2 by OsARF12 mediates auxin and BR signaling. Our results reveal that the miR167-OsARF12 module works downstream of miR159 to regulate rice grain filling and grain size via OsCDKF;2 by controlling cell division and mediating auxin and BR signals.

Strigolactone and gibberellin signaling coordinately regulate metabolic adaptations to changes in nitrogen availability in rice.

Modern semi-dwarf rice varieties of the "Green Revolution" require a high supply of nitrogen (N) fertilizer to produce high yields. A better understanding of the interplay between N metabolism and plant developmental processes is required for improved N-use efficiency and agricultural sustainability. Here, we show that strigolactones (SLs) modulate root metabolic and developmental adaptations to low N availability for ensuring efficient uptake and translocation of available N. The key repressor DWARF 53 (D53) of the SL signaling pathway interacts with the transcription factor GROWTH-REGULATING FACTOR 4 (GRF4) and prevents GRF4 from binding to its target gene promoters. N limitation induces the accumulation of SLs, which in turn promotes SL-mediated degradation of D53, leading to the release of GRF4 and thus promoting the expression of genes associated with N metabolism. N limitation also induces degradation of the DELLA protein SLENDER RICE 1 (SLR1) in an D14- and D53-dependent manner, effectively releasing GRF4 from competitive inhibition caused by SLR1. Collectively, our findings reveal a previously unrecognized mechanism underlying SL and gibberellin crosstalk in response to N availability, advancing our understanding of plant growth-metabolic coordination and facilitating the design of the strategies for improving N-use efficiency in high-yield crops.

OsCKX2 regulates phosphate deficiency tolerance by modulating cytokinin in rice.

Cytokinin oxidase/dehydrogenases (CKXs) are key enzymes that degrade cytokinins (CTKs) and play an essential role in plant growth and development. The present study analyzed the phenotypic and physiological characteristics of OsCKX2 overexpressing (OE) and knockout (KO) rice plants after exposure to phosphate (Pi) deficiency and the transcriptome and metabolome to investigate the function of OsCKX2 in response to Pi deficiency. OsCKX2 KO plants demonstrated higher endogenous CTK levels than wild-type (WT) under Pi deficiency. Further analysis indicated more robust tolerance of OsCKX2 KO plants to Pi deficiency, which exhibited higher phosphorus concentration, larger shoot biomass, and lesser leaf yellowing under Pi deficiency; whereas the opposite was observed for OsCKX2 OE plants. Transcriptome and metabolome analyses revealed that overexpression of OsCKX2 downregulated the transcriptional levels of genes related to Pi transporters, membrane lipid metabolism, and glycolysis, and reduced the consumption of metabolites in membrane lipid metabolism and glycolysis. On the contrary, knockout of OsCKX2 upregulated the expression of Pi transporters, and increased the consumption of metabolites in membrane lipid metabolism and glycolysis. These results indicated that OsCKX2 impacted Pi uptake, recycling, and plant growth via Pi transporters, phospholipid hydrolysis, and glycolysis under Pi deficiency. Overall, OsCKX2 negatively regulated Pi deficiency tolerance by modulating CTKs in rice.

Nitrate Modulates Lateral Root Formation by Regulating the Auxin Response and Transport in Rice.

Nitrate (NO3-) plays a pivotal role in stimulating lateral root (LR) formation and growth in plants. However, the role of NO3- in modulating rice LR formation and the signalling pathways involved in this process remain unclear. Phenotypic and genetic analyses of rice were used to explore the role of strigolactones (SLs) and auxin in NO3--modulated LR formation in rice. Compared with ammonium (NH4+), NO3- stimulated LR initiation due to higher short-term root IAA levels. However, this stimulation vanished after 7 d, and the LR density was reduced, in parallel with the auxin levels. Application of the exogenous auxin α-naphthylacetic acid to NH4+-treated rice plants promoted LR initiation to levels similar to those under NO3- at 7 d; conversely, the application of the SL analogue GR24 to NH4+-treated rice inhibited LR initiation to levels similar to those under NO3- supply by reducing the root auxin levels at 10 d. D10 and D14 mutations caused loss of sensitivity of the LR formation response to NO3-. The application of NO3- and GR24 downregulated the transcription of PIN-FORMED 2(PIN2), an auxin efflux carrier in roots. LR number and density in pin2 mutant lines were insensitive to NO3- treatment. These results indicate that NO3- modulates LR formation by affecting the auxin response and transport in rice, with the involvement of SLs.

SPL14/17 act downstream of strigolactone signalling to modulate rice root elongation in response to nitrate supply.

Nitrogen (N) is an essential major nutrient for food crops. Although ammonium (NH4+ ) is the primary N source of rice (Oryza sativa), nitrate (NO3- ) can also be absorbed and utilized. Rice responds to NO3- application by altering its root morphology, such as root elongation. Strigolactones (SLs) are important modulators of root length. However, the roles of SLs and their downstream genes in NO3- -induced root elongation remain unclear. Here, the levels of total N and SL (4-deoxyorobanchol) and the responses of seminal root (SR) lengths to NH4+ and NO3- were investigated in rice plants. NO3- promoted SR elongation, possibly due to short-term signal perception and long-term nutrient function. Compared with NH4+ conditions, higher SL signalling/levels and less D53 protein were recorded in roots of NO3- -treated rice plants. In contrast to wild-type plants, SR lengths of d mutants were less responsive to NO3- conditions, and application of rac-GR24 (SL analogue) restored SR length in d10 (SL biosynthesis mutant) but not in d3, d14, and d53 (SL-responsive mutants), suggesting that higher SL signalling/levels participate in NO3- -induced root elongation. D53 interacted with SPL17 and inhibited SPL17-mediated transactivation from the PIN1b promoter. Mutation of SPL14/17 and PIN1b caused insensitivity of the root elongation response to NO3- and rac-GR24 applications. Therefore, we conclude that perception of SLs by D14 leads to degradation of D53 via the proteasome system, which releases the suppression of SPL14/17-modulated transcription of PIN1b, resulting in root elongation under NO3- supply.

A Strigolactone Signal Inhibits Secondary Lateral Root Development in Rice.

Strigolactones (SLs) and their derivatives are plant hormones that have recently been identified as regulators of primary lateral root (LR) development. However, whether SLs mediate secondary LR production in rice (Oryza sativa L.), and how SLs and auxin interact in this process, remain unclear. In this study, the SL-deficient (dwarf10) and SL-insensitive (dwarf3) rice mutants and lines overexpressing OsPIN2 (OE) were used to investigate secondary LR development. The effects of exogenous GR24 (a synthetic SL analogue), 1-naphthylacetic acid (NAA; an exogenous auxin), 1-naphthylphthalamic acid (NPA; a polar auxin transport inhibitor), and abamine (a synthetic SL inhibitor) on rice secondary LR development were investigated. Rice d mutants with impaired SL biosynthesis and signaling exhibited increased secondary LR production compared with wild-type (WT) plants. Application of GR24 decreased the numbers of secondary LRs in dwarf10 (d10) plants but not in dwarf3 (d3), plants. These results indicate that SLs negatively regulate rice secondary LR production. Higher expression of DR5::GUS and more secondary LR primordia were found in the d mutants than in the WT plants. Exogenous NAA application increased expression of DR5::GUS in the WT, but had no effect on secondary LR formation. No secondary LRs were recorded in the OE lines, although DR5::GUS levels were higher than in the WT plants. However, on application of NPA, the numbers of secondary LRs were reduced in d10 and d3 mutants. Application of NAA increased the number of secondary LRs in the d mutants. GR24 eliminated the effect of NAA on secondary LR development in the d10, but not in the d3, mutants. These results demonstrate the importance of auxin in secondary LR formation, and that this process is inhibited by SLs via the D3 response pathway, but the interaction between auxin and SLs is complex.

Corrigendum: Nitric Oxide Affects Rice Root Growth by Regulating Auxin Transport Under Nitrate Supply.

[This corrects the article DOI: 10.3389/fpls.2018.00659.].

Overexpression of OsPIN2 Regulates Root Growth and Formation in Response to Phosphate Deficiency in Rice.

The response of root architecture to phosphate (P) deficiency is critical in plant growth and development. Auxin is a key regulator of plant root growth in response to P deficiency, but the underlying mechanisms are unclear. In this study, phenotypic and genetic analyses were undertaken to explore the role of OsPIN2, an auxin efflux transporter, in regulating the growth and development of rice roots under normal nutrition condition (control) and low-phosphate condition (LP). Higher expression of OsPIN2 was observed in rice plants under LP compared to the control. Meanwhile, the auxin levels of roots were increased under LP relative to control condition in wild-type (WT) plants. Compared to WT plants, two overexpression (OE) lines had higher auxin levels in the roots under control and LP. LP led to increased seminal roots (SRs) length and the root hairs (RHs) density, but decreased lateral roots (LRs) density in WT plants. However, overexpression of OsPIN2 caused a loss of sensitivity in the root response to P deficiency. The OE lines had a shorter SR length, lower LR density, and greater RH density than WT plants under control. However, the LR and RH densities in the OE lines were similar to those in WT plants under LP. Compared to WT plants, overexpression of OsPIN2 had a shorter root length through decreased root cell elongation under control and LP. Surprisingly, overexpression of OsPIN2 might increase auxin distribution in epidermis of root, resulting in greater RH formation but less LR development in OE plants than in WT plants in the control condition but levels similar of these under LP. These results suggest that higher OsPIN2 expression regulates rice root growth and development maybe by changing auxin distribution in roots under LP condition.

High temperature and drought stress cause abscisic acid and reactive oxygen species accumulation and suppress seed germination growth in rice.

Seed germination is one of the most important biological processes in the life cycle of plants, and temperature and water are the two most critical environmental factors that influence seed germination. In the present study, we investigated the roles of the plant hormone abscisic acid (ABA) and reactive oxygen species (ROS) in high temperature (HT) and drought-induced inhibition of rice seed germination. HT and drought stress caused ABA accumulation in seeds and inhibited seed germination and seedling establishment. Quantitative real-time polymerase chain reaction analysis revealed that HT and drought stress induced the expression of OsNCED3, a key gene in ABA synthesis in rice seeds. In addition, ROS (O2•- and H2O2) and malondialdehyde contents were increased in germinating seeds under HT and drought stress. Moreover, we adopted the non-invasive micro-test technique to detect H2O2 and Ca2+ fluxes at the site of coleoptile emergence. HT and drought stress resulted in a H2O2 efflux, but only drought stress significantly induced Ca2+ influx. Antioxidant enzyme assays revealed that superoxide dismutase (SOD), peroxidase, catalase (CAT), and ascorbate peroxidase (APX) activity were reduced by HT and drought stress, consistent with the expression of OsCu/ZnSOD, OsCATc, and OsAPX2 during seed germination. Altogether, these results suggest that ABA and ROS accumulation under HT and drought conditions can inhibit rice seed germination and growth.

A Transcription Factor, OsMADS57, Regulates Long-Distance Nitrate Transport and Root Elongation.

Root nitrate uptake adjusts to the plant's nitrogen demand for growth. Here, we report that OsMADS57, a MADS-box transcription factor, modulates nitrate translocation from rice (Oryza sativa) roots to shoots under low-nitrate conditions. OsMADS57 is abundantly expressed in xylem parenchyma cells of root stele and is induced by nitrate. Compared with wild-type rice plants supplied with 0.2 mM nitrate, osmads57 mutants had 31% less xylem loading of nitrate, while overexpression lines had 2-fold higher levels. Shoot-root 15N content ratios were 40% lower in the mutants and 76% higher in the overexpression lines. Rapid NO3 - root influx experiments showed that mutation of OsMADS57 did not affect root nitrate uptake. Reverse transcription quantitative PCR analysis of OsNRT2 nitrate transporter genes showed that after 5 min in 0.2 mM nitrate, only OsNRT2.3a (a vascular-specific high-affinity nitrate transporter) had reduced (by two-thirds) expression levels. At 60 min of nitrate treatment, lower expression levels were also observed for three additional NRT2 genes (OsNRT2.1/2.2/2.4). Conversely, in the overexpression lines, four NRT2 genes had much higher expression profiles at all time points tested. As previously reported, OsNRT2.3a functions in nitrate translocation, indicating the possible interaction between OsMADS57 and OsNRT2.3a Yeast one-hybrid and transient expression assays demonstrated that OsMADS57 binds to the CArG motif (CATTTTATAG) within the OsNRT2.3a promoter. Moreover, seminal root elongation was inhibited in osmads57 mutants, which may be associated with higher auxin levels in and auxin polar transport to root tips of mutant plants. Taken together, these results suggest that OsMADS57 has a role in regulating nitrate translocation from root to shoot via OsNRT2.3a.

The interaction between miR160 and miR165/166 in the control of leaf development and drought tolerance in Arabidopsis.

MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in plant development and abiotic stresses. To date, studies have mainly focused on the roles of individual miRNAs, however, a few have addressed the interactions among multiple miRNAs. In this study, we investigated the interplay and regulatory circuit between miR160 and miR165/166 and its effect on leaf development and drought tolerance in Arabidopsis using Short Tandem Target Mimic (STTM). By crossing STTM160 Arabidopsis with STTM165/166, we successfully generated a double mutant of miR160 and miR165/166. The double mutant plants exhibited a series of compromised phenotypes in leaf development and drought tolerance in comparison to phenotypic alterations in the single STTM lines. RNA-seq and qRT-PCR analyses suggested that the expression levels of auxin and ABA signaling genes in the STTM-directed double mutant were compromised compared to the two single mutants. Our results also suggested that miR160-directed regulation of auxin response factors (ARFs) contribute to leaf development via auxin signaling genes, whereas miR165/166- mediated HD-ZIP IIIs regulation confers drought tolerance through ABA signaling. Our studies further indicated that ARFs and HD-ZIP IIIs may play opposite roles in the regulation of leaf development and drought tolerance that can be further applied to other crops for agronomic traits improvement.

OsPIN1b is Involved in Rice Seminal Root Elongation by Regulating Root Apical Meristem Activity in Response to Low Nitrogen and Phosphate.

The response of plant root development to nutrient deficiencies is critical for crop production. Auxin, nitric oxide (NO), and strigolactones (SLs) are important regulators of root growth under low-nitrogen and -phosphate (LN and LP) conditions. Polar auxin transport in plants, which is mainly dependent on auxin efflux protein PINs, creates local auxin maxima to form the basis for root initiation and elongation; however, the PIN genes that play an important role in LN- and LP-modulated root growth remain unclear. qRT-PCR analysis of OsPIN family genes showed that the expression of OsPIN1b is most abundant in root tip and is significantly downregulated by LN, LP, sodium nitroprusside (SNP, NO donor), and GR24 (analogue of SLs) treatments. Seminal roots in ospin1b mutants were shorter than those of the wild type; and the seminal root, [3H]IAA transport, and IAA concentration responses to LN, LP, SNP, and GR24 application were attenuated in ospin1b-1 mutants. pCYCB1;1::GUS expression was upregulated by LN, LP, SNP, and GR24 treatments in wild type, but not in the ospin1b-1 mutant, suggesting that OsPIN1b is involved in auxin transport and acts as a downstream mediator of NO and SLs to induce meristem activity in root tip in rice under LN and LP.

Nitric Oxide Affects Rice Root Growth by Regulating Auxin Transport Under Nitrate Supply.

Nitrogen (N) is a major essential nutrient for plant growth, and rice is an important food crop globally. Although ammonium (NH4+) is the main N source for rice, nitrate (NO3-) is also absorbed and utilized. Rice responds to NO3- supply by changing root morphology. However, the mechanisms of rice root growth and formation under NO3- supply are unclear. Nitric oxide (NO) and auxin are important regulators of root growth and development under NO3- supply. How the interactions between NO and auxin in regulating root growth in response to NO3- are unknown. In this study, the levels of indole-3-acetic acid (IAA) and NO in roots, and the responses of lateral roots (LRs) and seminal roots (SRs) to NH4+ and NO3-, were investigated using wild-type (WT) rice, as well as osnia2 and ospin1b mutants. NO3- supply promoted LR formation and SR elongation. The effects of NO donor and NO inhibitor/scavenger supply on NO levels and the root morphology of WT and nia2 mutants under NH4+ or NO3- suggest that NO3--induced NO is generated by the nitrate reductase (NR) pathway rather than the NO synthase (NOS)-like pathway. IAA levels, [3H] IAA transport, and PIN gene expression in roots were enhanced under NO3- relative to NH4+ supply. These results suggest that NO3- regulates auxin transport in roots. Application of SNP under NH4+ supply, or of cPTIO under NO3- supply, resulted in auxin levels in roots similar to those under NO3- and NH4+ supply, respectively. Compared to WT, the roots of the ospin1b mutant had lower auxin levels, fewer LRs, and shorter SRs. Thus, NO affects root growth by regulating auxin transport in response to NO3-. Overall, our findings suggest that NO3- influences LR formation and SR elongation by regulating auxin transport via a mechanism involving NO.

A sensitive synthetic reporter for visualizing cytokinin signaling output in rice.

BACKGROUND: Cytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear. RESULTS: Early-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs, mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn. TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing. CONCLUSIONS: We report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.

Multiple roles of nitric oxide in root development and nitrogen uptake.

Nitric oxide (NO) is widely recognized for its role as a signaling molecule in regulating plant developmental processes. We summarize recent work on NO generation via nitrate reductase (NR) or/and NO synthase (NOS) pathway in response to nutrient fluctuation and its regulation of plant root growth and N metabolism. The promotion or inhibition of root development most likely depends on NO concentrations and/or experimental conditions. NO plays an important role in regulating plant NR activity at posttranslational level probably via a direct interaction mechanism, thus contributing largely to N assimilation. NO also regulates N distribution and uptake in many plant species. In rice cultivar, NR-generated NO plays a pivotal role in improving N uptake capacity by increasing root growth and inorganic N uptake, representing a potential strategy for rice adaption to a fluctuating nitrate supply.

The Interaction between Auxin and Nitric Oxide Regulates Root Growth in Response to Iron Deficiency in Rice.

Fe deficiency (-Fe) is a common abiotic stress that affects the root development of plants. Auxin and nitric oxide (NO) are key regulator of root growth under -Fe. However, the interactions between auxin and NO regulate root growth in response to Fe deficiency are complex and unclear. In this study, the indole-3-acetic acid (IAA) and NO levels in roots, and the responses of root growth in rice to different levels of Fe supply were investigated using wild type (WT), ospin1b and osnia2 mutants. -Fe promoted LR formation but inhibited seminal root elongation. IAA levels, [3H] IAA transport, and expression levels of PIN1a-c genes in roots were reduced under -Fe, suggesting that polar auxin transport from shoots to roots was decreased. Application of IAA to -Fe seedlings restored seminal root length, but not LR density, to levels similar to those under normal Fe (+Fe), and the seminal root length was shorter in two ospin1b mutants relative to WT under +Fe, but not under -Fe, confirming that auxin transport participates in -Fe-inhibited seminal root elongation. Moreover, -Fe-induced LR density and -Fe-inhibited seminal root elongation paralleled NO production in roots. Interestingly, similar NO accumulation and responses of LR density and root elongation were observed in osnia2 mutants compared to WT, and the higher expression of NOA gene under -Fe, suggesting that -Fe-induced NO was generated via the NO synthase-like pathway rather than the nitrate reductase pathway. However, IAA could restore the functions of NO in inhibiting seminal root elongation, but did not replace the role of NO-induced LR formation under -Fe. Overall, our findings suggested that NO functions downstream of auxin in regulating LR formation; NO-inhibited seminal root elongation by decreasing meristem activity in root tips under -Fe, with the involvement of auxin.