MicroRNAs (miRNAs) play key roles in plant growth, development and responses to abiotic stress. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants. However, it is yet unknown how miRNAs work during PAH uptake by plant roots. Thus, in this study we ascertain phenanthrene (a model PAH)-responsive miRNAs using small RNA high-throughput deep sequencing and their target genes in wheat roots. We identified 108 conserved and non-conserved miRNA members belonging to 82 miRNA families and found 11 differentially expressed miRNAs, among which four miRNAs (miR156, miR164, miR171a and miR9678-3p) were up-regulated and the other seven miRNAs (miR398, miR531, miR1121, miR5048-5p, miR9653b, miR9773 and miR9778) were down-regulated. ABC-transporter-related Gene CA704421 and CA697226 did not respond to phenanthrene exposure. miR156 and miR164 might regulate directly the growth and development of wheat roots by targeting SPL and NAC, respectively. miR398 and miR1121 could regulate oxidative reactions to respond to phenanthrene stress. Additionally, miR9773 might involve phenanthrene metabolism through acting on CYP450. Therefore, it is concluded that phenanthrene triggers variation in miRNA expression, which is associated with uptake of and response to phenanthrene. These findings are of significance for further understanding miRNA regulation mechanisms on PAH uptake, and providing guidance for screening of resistant cultivars in crop production and phytoremediation of PAH-contaminated soils or water at genetic level.

Phosphorus is one of the macronutrients essential for plant growth and development. The acquisition and translocation of phosphate are pivotal processes of plant growth. In a large number of plants, phosphate uptake by roots and translocation within the plant are presumed to occur via a phosphate/proton cotransport mechanism.

Members of the Low Phosphate Root (LPR) family have been identified in rice (Oryza sativa) and expression analyses have been conducted. Here, we investigated the functions of one of the five members in rice, LPR5. qRT-PCR and promoter-GUS reporter analyses indicated that under Pi-sufficient conditions OsLPR5 was highly expressed in the roots, and specific expression occurred in the leaf collars and nodes, and its expression was increased under Pi-deficient conditions. In vitro analysis of the purified OsLPR5 protein showed that it exhibited ferroxidase activity. Overexpression of OsLPR5 triggered higher ferroxidase activity, and elevated concentrations of Fe(III) in the xylem sap and of total Fe in the roots and shoots. Transient expression of OsLPR5 in Nicotiana benthamiana provided evidence of its subcellular localization to the cell wall and endoplasmic reticulum. Knockout mutation in OsLPR5 by means of CRISPR-Cas9 resulted in adverse effects on Pi translocation, on the relative expression of Cis-NATOsPHO1;2, and on several morphological traits, including root development and yield potential. Our results indicate that ferroxidase-dependent OsLPR5 has both a broad-spectrum influence on growth and development in rice as well as affecting a subset of physiological and molecular traits that govern Pi homeostasis.

In wetland soils, changes in oxygen (O2) level in the rhizosphere are believed to influence the behaviour of nutrients and their usage by plants. However, the effect of aeration on nitrogen (N) acquisition under different N supply conditions remains largely unknown. In this study, the rice cultivars Yangdao 6 (YD6, with higher root aerenchyma abundance) and Nongken 57 (NK57, with lower root aerenchyma abundance) were used to evaluate the effects of aeration on rice growth and N accumulation. Our results showed that the number of adventitious roots and the root surface area increased significantly, and ethylene production and aerenchyma formation decreased in both cultivars after external aeration (EA). Five N treatments, including no N (-N), 0.125 mM NH4NO3 (LN), 1.25 mM Ca(NO3)2 (NO3-N), 1.25 mM (NH4)2SO4 (NH4-N) and 1.25 mM NH4NO3 (N/N), were applied to YD6 and NK57 for 2 days under internal aeration or EA conditions. External aeration increased the root biomass in both cultivars and the shoot biomass in NK57 by 18-50 %. The total N concentrations in roots of YD6 grown under -N and LN and of NK57 grown under NO3-N were increased by EA. Expression of OsPAD4, one of four putative genes regulating aerenchyma formation, showed a similar pattern alongside changes in the ethylene level in the EA-treated rice irrespective of the N treatments. Furthermore, expression of the high-affinity nitrate transporter gene OsNRT2.1 was increased by EA under -N, LN and NO3-N conditions. Our data provide evidence of an interaction between O2 and the supply of N in ethylene production, aerenchyma formation and N nutrition through modification of the expression of OsPAD4 and OsNRT2.1.

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.

Strigolactones (SLs) or their derivatives have recently been defined as novel phytohormones that regulate root development. However, it remains unclear whether SLs mediate root growth in response to phosphorus (P) and nitrogen (N) deficiency. In this study, the responses of root development in rice (Oryza sativa L.) to different levels of phosphate and nitrate supply were investigated using wild type (WT) and mutants defective in SL synthesis (d10 and d27) or insensitive to SL (d3). Reduced concentration of either phosphate or nitrate led to increased seminal root length and decreased lateral root density in WT. Limitation of either P or N stimulated SL production and enhanced expression of D10, D17, and D27 and suppressed expression of D3 and D14 in WT roots. Mutation of D10, D27, or D3 caused loss of sensitivity of root response to P and N deficiency. Application of the SL analogue GR24 restored seminal root length and lateral root density in WT and d10 and d27 mutants but not in the d3 mutant, suggesting that SLs were induced by nutrient-limiting conditions and led to changes in rice root growth via D3. Moreover, P or N deficiency or GR24 application reduced the transport of radiolabelled indole-3-acetic acid and the activity of DR5::GUS auxin reporter in WT and d10 and d27 mutants. These findings highlight the role of SLs in regulating rice root development under phosphate and nitrate limitation. The mechanisms underlying this regulatory role involve D3 and modulation of auxin transport from shoots to roots.

Plant HAK/KUP/KT family members function as plasma membrane (PM) H+/K+ symporters and may modulate chemiosmotically-driven polar auxin transport (PAT). Here, we show that inactivation of OsHAK5, a rice K+ transporter gene, decreased rootward and shootward PAT, tiller number, and the length of both lateral roots and root hairs, while OsHAK5 overexpression increased PAT, tiller number, and root hair length, irrespective of the K+ supply. Inhibitors of ATP-binding-cassette type-B transporters, NPA and BUM, abolished the OsHAK5-overexpression effect on PAT. The mechanistic basis of these changes included the OsHAK5-mediated decrease of transmembrane potential (depolarization), increase of extracellular pH, and increase of PM-ATPase activity. These findings highlight the dual roles of OsHAK5 in altering cellular chemiosmotic gradients (generated continuously by PM H+-ATPase) and regulating ATP-dependent auxin transport. Both functions may underlie the prominent effect of OsHAK5 on rice architecture, which may be exploited in the future to increase crop yield via genetic manipulations.

OsSIZ1, a small ubiquitin-related modifier (SUMO) E3 ligase, exerts regulatory influences on the developmental responses and phosphate (Pi) homeostasis in rice (Oryza sativa). Whether paralogs OsSIZ1 and OsSIZ2 are functionally redundant or the latter regulates these traits independent of the former is not known. To determine this, in this study, OsSIZ2 was functionally characterized by employing reverse genetic approaches. Although the relative expression of OsSIZ2 was spatiotemporally regulated, it showed constitutive expression in root and leaf blade irrespective of Pi regime. Analysis of T-DNA insertion knockout (ossiz2) and RNAi-mediated knockdown (Ri1-3) mutants revealed positive influences on growth and developmental responses including yield-related traits. On the contrary, these mutants exhibited negative effects on the concentrations of Pi and total P in different tissues. The relative expression levels of some of the genes that are involved in Pi sensing and signaling cascades were differentially modulated in the mutants. Further, attenuation in the expression levels of OsSIZ2 in the roots of ossiz1 and relatively similar trend of the effects of the mutation in OsSIZ1 and OsSIZ2 on growth and development and total P concentration in different tissues suggested a prevalence of partial functional redundancy between these paralogs.

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.

Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus Rhizophagus irregularis remarkably promoted rice (Oryza sativa) growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO3- supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene OsNPF4.5 in rice roots, and its orthologs ZmNPF4.5 in Zea mays and SbNPF4.5 in Sorghum bicolor OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO3- transport activity when expressed in Xenopus laevis oocytes. Moreover, knockout of OsNPF4.5 resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO3- was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO3- acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.

Nitrogen (N) and carbon (C) are essential elements for plant growth and crop yield. Thus, improved N and C utilisation contributes to agricultural productivity and reduces the need for fertilisation. In the present study, we find that overexpression of a single rice gene, Oryza sativa plasma membrane (PM) H+-ATPase 1 (OSA1), facilitates ammonium absorption and assimilation in roots and enhanced light-induced stomatal opening with higher photosynthesis rate in leaves. As a result, OSA1 overexpression in rice plants causes a 33% increase in grain yield and a 46% increase in N use efficiency overall. As PM H+-ATPase is highly conserved in plants, these findings indicate that the manipulation of PM H+-ATPase could cooperatively improve N and C utilisation, potentially providing a vital tool for food security and sustainable agriculture.

Phosphorus (P) deficiency is one of the major nutrient stresses limiting plant growth. The uptake of P by plants is well considered to be mediated by a number of high-affinity phosphate (Pi) transporters belonging to the Pht1 family. Although the Pht1 genes have been extensively identified in several plant species, there is a lack of systematic analysis of the Pht1 gene family in any solanaceous species thus far.

Plant Phosphate Transporter 1 (PHT1) proteins, probably the only influx transporters for phosphate (Pi) uptake, are partially degraded on sufficient Pi levels to prevent excessive Pi accumulation. Therefore, the basal/constitutive expression level of PHT1 genes is vital for maintaining Pi uptake under Pi-replete conditions. Rice (Oryza sativa) OsPHT1;1 is a unique gene as it is highly expressed and not responsive to Pi, however the mechanism for maintaining its basal/constitutive expression remains unknown. Using biochemical and genetic approaches, we identified and functionally characterised the transcription factors maintaining the basal/constitutive expression of OsPHT1;1. OsWRKY21 and OsWRKY108 interact within the nucleus and both bind to the W-box in the OsPHT1;1 promoter. Overexpression of OsWRKY21 or OsWRKY108 led to increased Pi accumulation, resulting from elevated expression of OsPHT1;1. By contrast, oswrky21 oswrky108 double mutants showed decreased Pi accumulation and OsPHT1;1 expression in a Pi-dependent manner. Moreover, similar to ospht1;1 mutants, plants expressing the OsWRKY21-SRDX fusion protein (a chimeric dominant suppressor) were impaired in Pi accumulation in Pi-replete roots, accompanied by downregulation of OsPHT1;1 expression. Our findings demonstrated that rice WRKY transcription factors function redundantly to promote Pi uptake by activating OsPHT1;1 expression under Pi-replete conditions, and represent a novel pathway independent of the central Pi signalling system.