A new insight into root responses to external cues: Paradigm shift in nutrient sensing

Abstract

Higher plants are sessile and their growth relies on nutrients present in the soil. The acquisition of nutrients is challenging for plants. Phosphate and nitrate sensing and signaling cascades play significant role during adverse conditions of nutrient unavailability. Therefore, it is important to dissect the mechanism by which plant roots acquire nutrients from the soil. Root system architecture (RSA) exhibits extensive developmental flexibility and changes during nutrient stress conditions. Growth of root system in response to external concentration of nutrients is a joint operation of sensor or receptor proteins along with several other cytoplasmic accessory proteins. After nutrient sensing, sensor proteins start the cellular relay involving transcription factors, kinases, ubiquitin ligases and miRNA. The complexity of nutrient sensing is still nebulous and many new players need to be better studied. This review presents a survey of recent paradigm shift in the advancements in nutrient sensing in relation to plant roots.

Introduction

Nutrient elements are needed by plants for the successful completion of their life cycles. Worldwide, the demand of chemical fertilizers is increasing at an unprecedented rate. Therefore, instead of fertilizing the soil and destroying the natural environment of rhizosphere it would be prudent to reverse the excessive usage of chemical fertilization of soil. There is a pressing need to look for alternatives for chemical fertilizers such as biofertlisers and organic farming.¹ Additionally, methods such as mining of the nutrient induced genes and stress related genes that influence root architecture are also needed. However, deciphering the sensing and signaling cascades that are triggered in response to nutrient deficiencies in soils remains a major challenge. Roots provide anchorage, interact with biotic factors like mycorrhiza and soil dwelling organisms, uptake and absorb nutrients and water from rhizosphere. The root system is programmed to respond to varying concentrations of nutrients in soils and helps plants to adapt to nutrients scarcities.² Once an area sufficient in nutrients is sensed through the particular receptor on the roots, the internal signal transduction cascade comes into existence to allow favorable events to occur that induce root growth in the nutrient rich area.

However, the exact mechanism of sensing through the receptors is yet to be discovered. Various transporter proteins and transcription factors that are involved in signaling related to the transport of mineral ions across the root cell are sparsely known. Changes in nutrients availability also regulate alterations in the concentration of various phytohormones.³ Specifically auxin that has been already reported to be involved in root development.⁴ Other phytohormones such as ABA, ethylene and strigolactones have also been implicated in orchestrating nutrient deficiency-induced signaling related to root development.^2,5 The present review focuses on the cascade of events involved during phosphate (Pi) and nitrate (NO₃⁻) deficiencies.

Phosphate Starvation Induced Signaling

Phosphorous (P) plays a very significant role in the plant life cycle. It constitutes energy currency, ATP and nucleotide, the back-bone of DNA, phospholipids and also takes part in signal transduction pathway.⁶ Though P is abundant in the nature its absorbable form, inorganic phosphates (HPO₄²⁻ and H₂PO₄⁻; Pi), often remains unavailable and poses a real challenge to plants. Concentration of Pi in plant cell never exceeds 2 mM, whereas in soils its concentration never goes beyond 1 µM under natural agroclimatic conditions.⁷ Plant root system responds to changes in soil Pi concentration by stimulating its development toward the zones where the Pi pool is more abundant.^8,9 Pi (−Pi) induces inhibition in primary root growth in Arabidopsis thaliana⁹. Low Pi levels in soil causes reduction in cell differentiation in the meristem that can bring to the exhaustion of the mitotic activity.¹⁰

Genes Involved in Phosphate Induced Nutrient Sensing

There are many genes that showed up regulation during Pi deficient conditions. For example in Arabidopsis, 612 genes were upregulated whereas 254 downregulated during low Pi conditions.⁶ Low Pi induced genes in yeast come under the category of PHO-regulon. PHO genes encode acidic and alkaline phosphatases (PHO5, PHO8, PHO10, and PHO11) and negative regulators (PHO80, PHO85) that act under sufficient concentration of phosphates¹¹ (Fig.1). Interestingly, PHO-regulon genes in yeast showed homology with the cis-elements of tomato TPSI1 and Medicago truncatula Mt4 (cDNA representing a phosphate-starvation-inducible gene from Medicago truncatula) promoter sequences.^12,13 The PHO-like (CBKGTGG) and the TATA box-like (TATAAATA) elements have been noticed in the promoter of early response genes whereas another cis-element (CGCATATTCC), which is a PHR1 binding site, has been found in the late starvation induced genes.^13,14 Several phosphate starvation responsive genes, PSR, have been identified during the last decades in plants, namely rice and Arabidopsis, using large scale transcriptomic analysis.¹⁵ Several secreted purple acid phosphatases (PAPs) such as AtPAP10, AtPAP12 or AtPAP26, PvPAP1 and PvPAP3 have been shown to cause release of Pi organic P forms like ADP, glycerol-3-P or DNA in Arabidopsis and dNTPs in common bean (Phaseolus vulgaris).¹⁶ Also release of H⁺ and organic acids has also been shown to activate the insoluble inorganic P from soils¹⁷ Tomato 14-3-3, a protein, which functions as H⁺-ATPase has been suggested to provide H⁺ under low P conditions.¹⁸

Figure 1.

Schematic representation of nutrient and stress sensing by root cell. An unknown receptor sends the message to the unknown proteins which in connection with CBL/CIPK induces the genes responsible for auxin signaling. Similarly other mechanism also works along with CBL/CIPK pathway which induces various auxin, ethylene, gibberellin, phosphate induced genes. Several transcription factors, TFs are also induced due to phosphate deficiency which further control various genes. LBD37/38/39-lateral organ boundary domain, ARF- Auxin response factor 19, AFB3-Auxin signaling f-box 3, DGDG-Digalactosyldiacylglycerol, SQDG -Sulfoquinovosyl diacylglycerol, LPI- (Low Phosphate Insensitive), LPR1-1 (Low Phosphate Responsive), PSI-(Phosphate Starvation Insensitive, PDR2- Phosphate Deficiency Response 2, OSPHR1-Phosphate Starvation Response 1, SPL9- Squamosa Promoter Binding Protein 9, MYB62-MYB Binding Domain 62), ZAT6-Zinc Finger OF Arabidopsis 6, BHLH32-Basic Helix-Loop-Helix, PRD-Phosphate Root Development, HRS1-To Low Phosphate-Elicited Primary Root Shortening 1, ERF070-Ethylene Response Factor 070, PHR1 (Phosphate Starvation Response Regulator 1), MIR399- MicroRNA399, NAP1;2 -Nucleosome Assembly Protein, HRS1- To Low Pi-Elicited Primary Root Shortening 1; TIR1-Transport Inhibitor Response 1, GRF-General Response Factor, LEPT- Lycopersicon Phosphate Transporter, GLPT-Glycerol-3-Phosphate Transporter, IPS1- Pi Starvation-Induced Gene, RNS1-Arabidopsis Ribonuclease Gene, SIZ-SUMO/SMT3 Ligase, PSTOL -Phosphorous-Starvation Tolerance1 PSTOL, HPS2/3-Hypersensitive To Pi Starvation 2, SOC1-Suppressor Of Overexpression Of Constans1, CDKA-A-Type Cyclin-Dependent Kinase, CYCD3-Arabidopsis Cyclin D3, NPL7- Nodule Inception-Like Protein 7., CBL- Calcineurin B-Like (CBL) Proteins, CIPK- CBL Interacting Protein Kinases. NRT- Nitrate Transporter, AMT- Ammonium Transporter, NPF- Nitrate Transporter 1/Peptide Transporter Family.

Several transcription factors such as MYB62 (Myb binding domain 62), ZAT6 (Zinc finger of Arabidopsis 6), WRKY6, WRKY75, OsPTF1/ZmPTF1, bHLH32 (basic Helix-Loop-Helix), PRD (Phosphate Root Development), HRS1 (To low phosphate-elicited primary root shortening 1), and ERF070 (Ethylene response factor 070) have been identified from Arabidopsis in response to Pi deprivation.^19,20 Similarly, PHR1 (Phosphate starvation response regulator 1), a conserved MYB transcription factor that showed homology with PSR1 of Chlamydomonas, has been reported to regulate many Pi responsive genes and microRNA399 (miR399) family members.²¹ Interestingly, microRNA399 provides a long distance mobile signal during Pi homeostasis that travels through phloem.^22,23,24 PHO2 expression is dependent on microRNA399 and the binding site of miRNA is present on the 5′ UTR of PHO2. Both PHO2 and micro RNA399 showed co-regulation during Pi deficient conditions.²⁵ Expression of miR399 in shoots has been suggested to be linked by AM infestation.²⁶ Following the unknown mechanism it levels increases in the shoots with the entry of the AM through roots.²⁴ Increased levels of miR399 have been shown to prevent the expression of MtPho2 which suppresses Pi uptake in the roots.²⁶ In fact, ectopic expression of miR399s caused enhanced Pi uptake and root-to-shoot translocation in Arabidopsis plants.²⁷ MiR399 has also been reported to repress the transcripts of PHO2 and NLA²⁸ (Fig. 1).

Pi-H⁺ Co-transporters, PHT, have been shown to provide Pi to the plant roots from the rhizosphere.²⁹ Wheat and common bean PHT promoters have PHR binding sequences which mean PHR regulates the activity of PHT.²⁹ Activity of PHTs is regulated by PHR which Phr1 mutants have been shown to accumulate less anthocyanin, and they have reduced root–shoot ratio in comparison with wild type.^30,31 Similarly, rice OsPHR1 and OsPHR2 have been found to be homologous to Arabidopsis PHR1.^15,32,33 OsPHR1 and OsPHR2 were shown to be involved in Pi signaling by regulating the expression of genes that are involved in Pi-signaling pathway in rice.³⁴ Both OsPHR1 and OsPHR2 are nuclear proteins which are mostly expressed in roots and leaves.¹⁴ Similarly, other plants such as rice, wheat (TaPHR1) and common bean (PvPHR1) have similar proteins that help in the regulation of PHTs³⁵(Fig. 1).

GbWRKY1, a defense-related gene from cotton which is homologous to Arabidopsis AtWRKY75, has been shown to modify the root system in an auxin-dependent way.³⁶ Interestingly, overexpression of GbWRKY1 in the heterologous system Arabidopsis reduced symptoms of phosphate starvation.³⁶ Some of the low Pi responsive genes were also found to be involved in the local and systemic Pi sensing responses. Lpi (Low phosphate insensitive), lpr1-1 (Low phosphate responsive), and psi (Phosphate starvation insensitive) mutants have been found to show indeterminate primary root growth while pdr2 (Phosphate deficiency response 2) mutants have short root phenotype in response to low Pi.^37,38,39 Similarly, psi, and phosphate deficiency root hair defective1, per1 mutants have been found to have negative effect on the low Pi responsive root hair elongation³⁷ (Fig. 1).

Besides transcription factors, SIZ1, a SUMO E3 ligase (a suppressor of SOS3, salt over sensitive3) has also been reported to regulate root architecture response to Pi limitation giving tolerance against drought.^40,41 siz1 KO mutants exhibit enhanced typical P-starvation responses: arrest of primary root growth, extensive lateral root and root hair development, increased root/shoot ratio and greater anthocyanin accumulation.⁴² It has been shown that SIZ1 sumoylate PHR1 in vitro.⁴⁰ Interestingly in siz1 mutants the expression of the 2 Pi-starvation response genes, IPS1, a highly specific Pi starvation-induced gene, and RNS1 (Arabidopsis ribonuclease gene), which are regulated by PHR1, has been found to show slower induction than in wild type plants. Furthermore, it was revealed by microarray analysis that many auxin-responsive genes, such as genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 mutant plants in comparison to wild-type under Pi starvation.⁴⁰ Interestingly, mutation in MMS21 gene, methane sulfonate (MMS)-sensitive mutants, coding for the other SUMO E3 ligase in Arabidopsis, resulted in the inhibition of primary root while elongation and growth promotion was noticed in case of lateral roots.⁴² In tomato, LePT4, a phosphate transporter gene, has been shown to work under the regulation of low Pi levels and mycorrhizal colonization. Mutants of LePT4 showed stunted growth when grown under solution-Pi concentrations (Cp) of 0.05 mM and 0.5 mM and absence of mycorhizza in comparison to wild-type. Wild-type when subjected to mycorrhizal infection showed 2-fold increase in the Pi uptake when supplied with Cp of 0.05 mM whereas significantly lower level of Pi uptake was also observed in lept4 mutants. However, at Cp of 5 mM LePT4 mutant showed better growth in comparison to wild type.⁴³

Induction of high-affinity Pi transporters during Pi starvation plays an important role in the acquirement and mobilization of Pi in plants.⁴⁴ Similarly, glycerol-3-phosphate transporter (GlPt), a sugarphosphate/ anion antiporter, has also been reported to be involved in Pi mobilization.^45,46 There are ample reports of the presence of various genes that increase the absorption of phosphate during phosphate deficiency along with changing root architecture.⁹ However, the knowledge of nutrient sensing was further enhanced by the discovery of quantitative trait locus (QTL) for phosphorous-deficiency tolerance. PHO, one of the QTLs, has been reported from rice on the short arm of chromosome 12 flanked by markers RG9 and RG241, that is involved in phosphate-deficiency tolerance.^14,47 Four QTLs were detected for Pi uptake on chromosomes 2, 6, 9, and 10.^47,48,49

Pup1-specific protein kinase gene, which is also known as PHOSPHOROUS-STARVATION TOLERANCE1 (PSTOL1) was found to be absent in some of the intolerant varieties of rice that are susceptible to phosphorous starvation. This gene was also found to be an enhancer of early root growth. Interestingly, ectopic expression of PSTOL1 in susceptible varieties gave them high grain yield potential in phosphorus-deficient soil.⁵⁰ Similarly, a correlation between shoot traits and root morphologies in B. oleracea has been found when a QTL has been identified on chromosome C03 and C07 which is responsible for shoot-Pi and measures of Pi-use efficiency (PUE).⁵¹ Pht1;5, the inorganic phosphate (Pi) transporter gene has been reported to be involved in mobilising stored form of Pi from the older leaves to the places where they are required such as young leaves and roots specially under low Pi conditions. Interestingly, mutant of pht1;5 showed poor Pi allocation and upregulation of low Pi responsive genes.^52,53 Expression of phosphoserine-binding proteins, 14-3-3, has also been found to be regulated by Pi levels. 14-3-3 isoforms such as epsilon (GRF9, GRF10, GRF11, GRF13) and non-epsilon (GRF1, GRF3, GRF6, GRF8) (Fig. 1). Grf9 mutants showed increased accumulation of starch in the leaves and change in the pattern of root architecture under low Pi.⁵⁴ Lack of phosphate also influences the carbon metabolism that in turn reduces the concentration of phosphorylated sugar in comparison with its non-phosphorylated form. Activity of hexokinases and fructokinases has been shown to decrease during phosphate deficiency in the soil.^55,56 Glucose sensor HXK gene, when mutated in glucose insensitive mutant (gin2) has been shown to be insensitive toward auxin.⁵⁷ Both auxin and glucose regulates the rate of cell proliferation and expansion in roots.⁵⁸ Auxin plays a role in the development of lateral roots and is thus essential for proper root architecture.⁵⁹ Similarly, AtERF070, a Pi starvation-induced ethylene response factor belonging to AP2/ERF family of TFs in Arabidopsis has been shown to be localized in the nucleus. It is localized to the nucleus and has been shown to get induced specially during low Pi condition. Interestingly, AtERF070 silencing caused induction of lateral roots for the higher absorption of Pi whereas plant overexpressing AtERF070 showed suppression of both lateral and primary roots.⁶⁰ In addition, AtERF070 overexpressing and silenced plants showed differential expression of low Pi responsive genes⁶⁰ (Fig. 1).

There are some reports where the role of chromatin remodelling has been enumerated in low Pi related responses, the nuclear actin-related protein (APR6) and which is important part of SWR1 chromatin remodeling complex has been shown to regulate transcription via deposition of the H2A.Z at several low Pi responsive genes.⁶¹ Similarly, Pi responsive protein such as histone chaperone and nucleosome assembly protein, NAP1;2, have been identified through 2D gel electrophoresis and MALDI-TOF/TOF from the Pi starved Arabidopsis roots. NAP1;2 being a component of chromatin remodelling complex has been shown to play an important role during low Pi.⁶²

Hormonal Control of Phosphate Deficiency

Plant responses to phytohormones and (Pi) starvation are closely linked. However, the underlying mechanisms that connect hormone to phosphate starvation (−Pi) responses are mostly unclear. Phytohormones like auxin, gibberellins, cytokinin, and abscisic acid have been shown to play a role in root development. Auxin is synthesized in the young leaves and cotyledon and it moves from the phloem channel for the rapid transport.^63,64 Auxin transport generally takes place through polar auxin transport in concert with several membrane transport protein.^65,66

The modification of the root system architecture is closely associated with auxin signaling. Auxin plays a role in the development of lateral roots and is thus essential for proper root architecture.⁶⁷ Very recently, APSR1 (Altered Phosphate Starvation Response1) was reported as a transcription factor and found to be involved in the maintenance of the root meristem.^68,69 Interestingly apsr1 mutant in Arabidopsis resulted in reduced primary root length and root apical meristem size, short differentiated epidermal cells and long root hairs. A close link has been noticed between the auxin transporter PIN7 and APSR1. In fact, in the absence of ASPR1, PIN7 expression decreases under phosphate starvation conditions.⁶⁹ Similarly, an auxin response factor, OsARF16 has been shown to regulate auxin mediated root growth in response to -Pi level changes in rice (Oryza sativa L.). In Oryza sativa Auxin Response Factor16, OsARF16, mutants the root system has been found to be insensitive to auxin and -Pi response.⁷⁰ Expression of OsARF16 is induced by IAA and different Pi treatments.⁷¹ Six marker genes of Pi starvation were also shown to be repressed in osarf16 knock-out mutant.⁷¹ Overexpression of GbWRKY1 from Gossypium barbadense in Arabidopsis has been shown to enhance the sensitivity of roots to auxin, to increase the number of lateral roots, to increase the levels Pi in roots and to stimulate the accumulation of acid phosphatases even under sufficient phosphate level.³⁶ Moreover, the expression of 2 genes TIR1 and ARF19, which are involved in the regulation of the auxin sensitivity was induced by low Pi conditions and TIR1 expression was affected in GbWRKY1 overexpressors plants depending on the Pi conditions³⁶ (Fig. 1). Roles of Transporter Inhibitor Response, TIR1 and ARF19 (auxin response factor 19)-dependent auxin signaling have been already suggested in the regulation of lateral root development under Pi deficiency.⁷² Under low phosphate level the phosphorus starvation-insensitive (psi) mutant has been reported to show impaired accumulation of anthocyanin in leaves, reduction of primary root growth and root hair growth. The reported mutant was found to be defective in the LPR1 gene region. LPR1 gene has been reported to regulate auxin responses and to work independently of SUMO E3 ligase, SIZ1.⁷³

Genes involved in membrane lipid remodeling has been shown to get activated during phosphate deficiency.⁷⁴ Interestingly, it was found that phosphate starvation induces the synthesis of non-phosphorous and sulfoquinovosyl diacylglycerol (SQDG) by replacing phospholipids.⁷⁵ There are reports that prove the involvement of DGDG in the auxin transport. In both slr-1 and arf7-arf19 mutants Pi stress-induced accumulation of DGDG and SQDG is reduced.⁷⁶ Furthermore, regulation of glycolipid synthase and phospholipase genes was found to be reduced indicating the involvement of IAA14 and ARF7/19 in membrane lipid remodeling.^75,76 Cell cycle in the lateral roots of Arabidopsis is regulated by auxin.^77,78 Pi deficiency caused the regulation of cell cycle genes such as CDKA, E2Fa, Dp-E2F and CyCD3. However, E2Fa, Dp-E2F and CyCD3 genes are under the influence of auxin sensitivity in the roots and not on its transport^79,80 (Fig. 1). Furthermore, high expression of TIR1 causes higher degradation of AUX/IAA repressors which in turn allows ARF19 regulated cell cycle in the pericycle region⁸¹ (Fig. 1).

Collusion of sucrose and auxin in deciding the fate of root architecture is a matter of significance especially in the case of lateral root development. Under low Pi and sucrose conditions, auxin movement increases basipetally due to pyrophosphatase dependent apoplastic acidification.^60,82 The mechanism behind the change in fluxes as induced by low phosphate content is out of comprehension yet some evidences are suggested.⁸³ It has been shown that the sucrose and auxin signals interaction regulates the lateral and root hair development.⁸⁴ Moreover, the increase of lateral roots number in phosphate and sucrose defecient conditions in chalcone synthase tt4-2 mutant indicates a role of flavonoids in auxin-mediated modulation of RSA.⁸² Special endogenous compounds such as aglycone flavonols and strigolactones have been identified as regulators of polar auxin transport.^85,86,87 Genes that regulate flavonoid biosynthesis are induced by 2–3 folds under Pi starvation.⁸⁸ Though some contradictory results were also reported in which it was found that absence of flavonoids helped in the transport of auxin.⁸⁹

In one of the interesting reports it was found that GA was also found to influence signaling during Pi starvation through DELLA proteins.⁹⁰ DELLA is known to be negatively regulated by GA and GA level get reduced during Pi starvation.⁹⁰ DELLA-mediated signaling has been shown to increase the root hair length during Pi starvation.⁹¹ Moreover, some of the indicators of Pi starvation such as anthocyanin accumulation and changes in root architecture are influenced by DELLA-mediated signaling⁹¹ (Fig. 1). However, Pi starvation induced regulation of starvation responsive genes and Pi uptake efficiency remained unaffected.⁹¹ Similarly, the Pi deficiency responsive transcription factor MYB62 when over expressed has been shown to share similar phenotype with gibberellic acid (GA)-deficient mutants.⁹² However, several Pi starvation-induced genes are suppressed in MYB62 overexpressing plants.^60,92 This is the case of 2 flowering regulator genes, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and SUPERMAN that were also controlled by GA, whose expression is suppressed in MYB62 overexpressing plants, thus indicating the role of gibberellins signaling during Pi starvation.⁹² Like auxin and gibberellins, ethylene has also been found to influence root architecture during Pi starvation by inhibiting primary root growth and root hair formation.^93,94,95 Under Pi-starvation, the synthesis of ethylene increases in the roots of tomato and Medicago falcata plants.⁹⁶ Furthermore, Arabidopsis hps2 and hps3 (mutated in ETO1 -ETHYLENE OVERPRODUCTION 1-gene) mutants have been found to be involved in the marker effects of Pi starvation such as regulation of PSI gene expression and production of acid phosphatases and anthocyanin.^97,98

Overall, if movement and concentration of phytohormones is directing the root growth in response to nutrients then it is pertinent to explain how they act. Nevertheless, it would be interesting to interpret the role of various phytohormones by applying not only one but the mixture of various hormones with different concentrations.

Nitrate Responsive Signaling

Nitrogen (N) is another element that is required by plants in almost all physiological processes since it is a major constituent of DNA, amino acids and other complex molecules. Although abundant as a diatomic form (N₂) in the atmosphere, N availability is a major problem for plants because only a few species are able to indirectly assimilate N from N₂ through symbioses, whereas the vast majority of plants must acquire N from the inorganic and/or organic forms present in the soil. For most temperate herbaceous plants, nitrate (NO₃⁻) is the main source of N. Although highly soluble in water and thus readily available for plant roots from the soil, NO₃⁻ concentration in soils fluctuates dramatically as it is prone to leaching and consumed by most soil microorganisms in addition to plants despite its dynamic production by nitrifying bacteria. Plants need to constantly adapt to these fluctuations of external NO₃⁻ abundance to maintain optimal N acquisition. Therefore, they have developed sophisticated mechanisms for NO₃⁻ sensing and signaling, in order to quickly activate relevant responses. Like phosphate, acquisition of NO₃⁻ by plants is highly dependent on both the activity of specific transport proteins in root cells, and on the overall size and architecture of the root system. Accordingly, NO₃⁻ sensing and signaling pathways strongly regulate root NO₃⁻ transporters and root development.

Five NO₃⁻ transporters families namely, NRT2, SLAC/SLAH, ALMT, CIC and NPF (NRT1/PTR) have already been proposed to play a role in nitrogen sensing.⁹⁹ It has been shown that plants have 2 NO₃⁻ uptake systems: the High-Affinity transport System (HATS), and the Low-Affinity Transport System (LATS).^100,101 Nitrate is converted to nitrite by nitrate reductase encoded by NIA1 (nitrate reductase 1) and NIA2 (nitrate reductase 2) genes, and subsequently reduced to ammonia by nitrite reductase encoded by a single NIR gene.¹⁰² NPF6.3/NRT1.1 (formerly CHL1, CHLORATE RESISTANT MUTANT 1), NPF4.6/NRT1.2, NRT2.1, and NRT2.2, have been reported to transport NO₃⁻ from the external medium into the root cells,¹⁰³ while 4 other transporters, AMMONIUM TRANSPORTERS, AMT1.1, 1.2, 1.3, and 1.5) perform the same function with ammonium (NH₄⁺) as a substrate.¹⁰⁴ NRT1/PTR transporters belong to the NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY (NPF), which comprises 53 members in Arabidopsis.¹⁰⁵ NRT2 transporters belong to the NRT2/NNP (NITRATE NITRITE PORTER) family with 7 members in Arabidopsis (Krapp et al., 2014). NPF and NRT2 genes are not similar to each other on the basis of sequence homology.¹⁰⁶

NPF6.3/NRT1.1 has been reported to be a NO₃⁻ transporter induced by NO₃⁻, which acts as either HATS or LATS, in response to phosphorylation/dephosphorylation of the threonine 101 residue, respectively.¹⁰⁷ Indeed, it was shown in Xenopus oocytes that expression of the non-phosphorylable T101A mutant of NPF6.3/NRT1.1 leads to NO₃⁻ uptake in the oocytes only at high concentration, whereas, the phosphomimic T101D mutant triggers a lower NO₃⁻ influx capacity that saturates at low NO₃⁻ concentration. A NPF of Medicago truncatula (MtNRT1.3) that shows homology with Arabidopsis NPF6.4/NRT1.3, has also been identified as a dual-affinity NO₃⁻ transporter.¹⁰⁸ Furthermore, NPF6.3/NRT1.1 was recently reported to also mediate NO₃⁻ efflux out of the cells, and to contribute to NO₃⁻ translocation from root to shoot.¹⁰⁹ NPF4.6/NRT1.2 has been shown to function as a low-affinity NO₃⁻ transporter only.¹¹⁰ Located in the epidermis of roots, NPF4.6/NRT1.2 is constitutively expressed and thus corresponds to a LATS. The other NPFs characterized as NO₃⁻ transporters to date play other roles like uptake and distribution of NO₃⁻ in the shoot. For instance, NPF6.2/NRT1.4 is expressed in petioles, and thus to control NO₃⁻ accumulation in leaves.¹¹¹

Members of NRT2 family, such as NRT2.1, NRT2.2, and NRT2.4 stand among the components of the HATS.¹¹² NRT2 proteins harbor 12 predicted transmembrane domains. NRT2.1 in inducible by NO₃⁻, and is considered as the main component of the HATS, accounting for up to 75% of the high-affinity NO₃⁻ uptake by the plant.^113,114 In addition, NRT2.1 gene expression is feedback repressed in response to long-term supply of NO₃⁻, or to provision of the plant with reduced N sources such as NH₄⁺¹¹⁴.

The observation that the glutamate synthase inhibitor, azazerine is able to downregulate NRT2.1 has led to the conclusion that glutamine might be a specific repressor molecule for this gene.¹¹⁵ Furthermore, NRT2.1 expression is also stimulated by photosynthesis and sugars,¹¹⁶ indicating that this transporter plays a key role in controlling root NO₃⁻ uptake, not only because it is quantitatively the main contributor to this uptake, but also because it is the target of most regulatory mechanisms affecting root NO₃⁻ acquisition.¹¹⁷ Moreover, evidence is now accumulating that NRT2.1 is regulated at the posttranscriptional level in response to the same environmental factors that control expression of the gene.¹¹⁸ In plants as well as in green algae like Chlamydomonas reinhardtii, most NRT2 proteins need to interact with another protein called NAR2 to be functional.¹¹⁹ Arabidopsis has 2 NAR2 genes, NAR2.1/AtNRT3.1 and NAR2.2/AtNRT3.2, but only NAR2.1 has been shown to participate in high-affinity NO₃⁻ uptake.¹²⁰ The exact function of NAR2.1 is unknown. However, it is postulated that the actual NO₃⁻ transport system involving NRT2.1 is a hetero-oligomer containing 2NRT2.1/2NAR2.1.¹¹⁹

To date, nearly 20 NRT proteins are reported to be NO₃⁻ transporters in various organs and tissues of Arabidopsis,¹²¹ and it is likely that others remain to be identified. An additional complexity came into this picture after the finding that NO₃⁻ transporters may not only act as pure NO₃⁻ transporters, but are also putative NO₃⁻ sensors activating many adaptive responses of the plant to NO₃⁻.^{100,122,123,124} NRT2.1 controls root architecture through the regulation of the lateral root development.^124,125,126 The lin1 mutant that is deficient in the NRT2.1 gene showed derepression of lateral root development under high sucrose/low NO₃⁻ ratio.¹²⁷ However, under different environmental conditions, it was reported that NRT2.1 had at the opposite a stimulatory effect on lateral root development.¹²⁴ Despite these discrepancies, both studies agree that NRT2.1 seems to act specifically on the initiation of lateral root primordia even in the absence of NO₃⁻ in the external medium, suggesting that its role in regulating root development is not directly related to its NO₃⁻ transport function.^124,127 In Brassica napus, expression levels BnNRT2.1 have been shown to follow a linear pattern with the changes in root length.¹²⁸ The best case study highlighting a signaling role for a NO₃⁻ transporter is probably that of NPF6.3/NRT1.1.¹²⁹ Indeed, this protein is required for many different responses of the plant to NO₃⁻, including regulation of root development. Under low NO₃⁻ concentration, NPF6.3/NRT1.1 has been shown to repress emergence of lateral root primordia and growth of young lateral roots through the modification of auxin gradients in these organs.¹³⁰ This is expected to be due to the surprising capacity of NPF6.3/NRT1.1 to transport both NO₃⁻ and auxin.¹³⁰ and to explain at least in part the ability of plants to preferentially colonize NO₃⁻–rich patches of the soil.^123,131 In addition, NPF6.3/NRT1.1 triggers the NO₃⁻ induction of many NO₃⁻–responsive genes, including NRT2.1.¹²² In this case, the signaling role of NPF6.3/NRT1.1 is demonstrated by the fact that mutation of the proline 492 residue to leucine in the chl1–9 mutant suppresses the NO₃⁻ transport activity of the protein, without affecting the NO₃⁻ induction of NRT2.1.¹²²

In addition to NRT2.1 and NPF6.3/NRT1.1, at least 4 different classes of signaling proteins have been reported to contribute to NO₃⁻–induced responses. The first one relates to kinases belonging to the CBL-Interacting Protein Kinase (CIPK) family. Indeed, CIPK8 and CIPK23 work with NO₃⁻ signaling machinery to participate in the NO₃⁻ regulation of gene expression and/or of protein activity.¹³² CIPK8 and CIPK23 are themselves induced by NO₃⁻ and their induction are repressed in NPF6.3/NRT1.1 deficient mutants.¹³³ These two proteins physically interact with CBL (Calcineurin B-like protein) and CIPK8 is required for full NO₃⁻ induction of NRT2.1, and CIPK23 have been shown to phosphorylate NPF6.3/NRT1.1, at the T101 residue. The second category of regulatory proteins associated with NO₃⁻ signaling includes transcription factors. NIT2 has been shown to interact with Chlamydomonas NIA1 gene promoter to regulate intracellular nitrate by regulating the transcript levels of NIA1 gene.¹³⁴ NIT2 is homologous to Nin (for nodule inception) and its action has been shown to be negatively regulated by ammonium. Similarly, an Arabidopsis homolog of NIT2, NLP7 (Nodule Inception-Like protein 7), a RWP-RK domain–containing protein, has been also shown to play a similar role in the NO₃⁻ regulation of gene expression.^135,136 The mutant of this gene has been reported to be insensitive to NO₃⁻, shows altered responses to N starvation, and displays long primary root, increased lateral root density and reduced shoot-to-root biomass.¹³⁶ Also, mutant of npl7 mutant of Arabidopsis showed the phenotype similar to the N starved plant. Interestingly, it has been shown that npl7 repress genes that are induced during N deficient condition.¹³⁶ Overexpression of the SQUAMOSA PROMOTER BINDING PROTEIN 9 (SPL9) transcription factor, identified as a one of the early genes that respond to NO₃⁻, results in an increase in the induction of NiR gene by NO₃⁻, although the SPL9 deficient mutant does not show any obvious phenotype.¹³⁷ The three genes LBD37/38/39 belonging to LATERAL ORGAN BOUNDARY DOMAIN family are induced by NO₃⁻¹³⁸ (Fig. 1). These genes encode for DNA binding zinc finger transcription factors, and repress other NO₃⁻-induced genes such as NRT2 and NIA under high NO₃⁻ concentration. The MADS-box putative transcription factor ANR1 has also been reported to regulate root system architecture in response to NO₃⁻, likely downstream NPF6.3/NRT1.1.¹²⁴ In particular, ANR1 is suggested to play a key role in the preferential growth of lateral roots in NO₃⁻–rich patches of the external medium.¹³⁹ Recently, it was found that the GARP transcription factor HRS1 integrates nitrate and phosphate signals in the root. Indeed, HRS1 has a NO₃⁻/NLP7/CHL1 dependent transcriptional regulation and a Pi dependent post transcriptional regulation. The mutant hrs1 hho1 is impaired in the phosphate starvation dependent primary root shortening.¹⁴⁰ Finally, the third class of regulatory proteins contributing to the plant responses to NO₃⁻ are those associated with hormone signaling. For instance, under high NO₃⁻ supply, AUXIN RESPONSE FACTOR 8 (ARF8) has been shown to get activated in the pericycle of roots, which results in the induction of lateral root development.¹⁴¹ In addition, a regulatory module involving the auxin receptor AFB3 and the microRNA miR393 was shown to trigger several modifications of development in both primary and lateral roots¹⁴² (Fig. 1). However, though AFB3 is induced by NO₃⁻, the microRNA that targets AFB3 mRNA is activated by reduced nitrogen metabolites.¹⁴³ Auxin is by far not the only phytohormone involved, since NO₃⁻ has been reported to induce IPT3, encoding an adenosine phosphate-isopentenyltransferase (the first enzyme involved in cytokinin biosynthesis), which in turn increases the cytokinin levels in the xylem for translocation from roots to shoots.¹⁴⁴ Furthermore, cytokinins are suggested to play a major role in the long-distance signaling regulating NO₃⁻ uptake and assimilation.¹⁴⁵ The functions of various receptors and genes involved in nitrogen sensing are mostly unknown, although some genes have been identified but their actual role needs to be investigated. Finally, the ABA-regulated protein phosphatase 2C ABI2 is involved in nitrate transport and sensing.¹⁴⁶ ABI2 interacts with and inactivates CIPK23 and CBL1 leading to NPF6.3/NRT1.1 activation.¹⁴⁶

Summary and Outlook

The depletion of nutrients that are essential for plant growth and development is becoming a major problem and productivity is declining at an unprecedented rate. Our dependence on chemical fertilizers has encouraged the thriving of industries that are producing life-threatening chemicals which are not only hazardous for human consumption but can also disturb the ecological balance. Knowing the science behind nutrient sensing in plants can greatly contribute toward solving the problem of feeding an increasing global population at a time when agriculture is facing a grim challenge due to aridity and resultant nutrient deficiency in the soil. New and powerful tools provided by strides in molecular biotechnology can enhance the biological pathways which help plants in sensing even the small patch of nutrient abundance in the rhizosphere. However, the lack of knowledge regarding the sensing mechanism and inner sophisticated molecular machinery including transcription factors and responsible genes is one of the few reasons for the continuing monopoly of chemical fertilizers worldwide. Nevertheless, the recent advancements in the identification of sensors like NPF6.3/NRT1.1, associated transcription factors and kinases/phosphatase such as CIPK8, CIPK23 and ABI2 will help to dissect the pathways involved in nutrient sensing.^147,148 Receptors such as NPF6.3/NRT1.1, NPF4.6/NRT1.2, NRT2.1 & NRT2.2, AMT 1.1, 1.2, 1.3 and 1.5, can be suggested to be the potential receptors for the sensing of various nutrients (nitrate and ammonium) furthermore this sensing causes up-regulation and down-regulation of various genes under the control of phytohormones such as auxin, gibberellins and ethylene (Fig. 1). The success of the science related to nutrient sensing in plants depends on innovative strategies related to the functions of sensors and affected genes and their proper application to the field of agriculture. Nevertheless, the major challenge in this area of research will be to translate the useful knowledge of research done so far into improving the nutrient utilization capabilities of crops.

In conclusion, nutrient sensing in plant roots is a promising area of research that would help us to further understand the mechanisms of root growth in the context of nutrients present in the rhizosphere. Many receptors and genes that regulate the development of root architecture in the presence of nutrients have already been identified. However, stress conditions such as drought and aridity are adversely affecting soil quality and in turn crop production. This could be mitigated by the discovery of new genes that will help deciphering the mechanism of nutrient sensing and pathway related to root development. Present review summarizes the available contemporary knowledge on nutrient sensing and sheds lights on the role of various genes and transcription factors on root development and nutrient sensing.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are also thankful to Dr. Ananda K. Sarkar (NIPGR, New Delhi) and Dr. Ajay Jain (NRCPB, New Delhi) for their critical reading of the manuscript. AM is supported by a post-doctoral fellowship from Agence Nationale de la Recherche (ANR-JCJC “NUTSE” to BL).

Funding

BL and NT are thankful to Indo–French Center for the Promotion of Advanced Research (IFCPAR/CEFIPRA) for financial support of this work (Proposal No. 4609-A).