Journal of Genetics (2019) 98:74 © Indian Academy of Scienceshttps://doi.org/10.1007/s12041-019-1127-9

RESEARCH ARTICLE

Expression of the cassava nitrate transporter NRT2.1 enables Arabidopsislow nitrate tolerance

LIANGPING ZOU1, DENGFENG QI1, JIANBO SUN1, XU ZHENG2∗ and MING PENG1∗

1Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences,Haikou 571101, People’s Republic of China2Key Laboratory of Wheat and Maize Crop Science, College of Agronomy, Henan Agricultural University,Zhengzhou 450046, Henan, People’s Republic of China*For correspondence. E-mail: Xu Zheng, hello_zx@hotmail.com; Ming Peng, pengming@itbb.org.cn.

Received 1 April 2019; revised 28 May 2019; accepted 10 June 2019

Abstract. The cassava grows well on low-nutrient soils because of its high-affinity to absorb nitrate. However, the molecularmechanisms by which cassava adapts itself to this environment remain elusive, although we have cloned a putative gene namedMeNRT2.1which has a crucial role in high-affinity nitrate transporter from cassava seeding. Here, the expression pattern ofMeNRT2.1was further assessed using the GUS activity driven by MeNRT2.1 promoter in Arabidopsis transformation plants. The GUS activitywas monitored over time following the reduction of nitrate supply. The GUS gene expression not only peaked in roots after 12 h in0.2 mM nitrate media, but also stained stems and leaves. Arabidopsis plants with overexpression of MeNRT2.1 increased the biomasscompared to the wild type on rich nitrogen (N-full) media. However, chlorate sensitivity analysis showed that Arabidopsis plantsexpressing MeNRT2.1 were more susceptable to chlorate than wild type. Significantly, after growing for 15 days on media containing0.2 mM nitrate concentration, wild-type plants became yellow or died, while the transgenicMeNRT2.1 Arabidopsis plants maintainednormal growth. With significant increases in the amount of 15NO−

3 uptake in roots, the MeNRT2.1 plants also increased the contentsof chlorophyll and nitrate reductase. Taken together, these results demonstrate that MeNRT2.1 has an important role in adaptationto low nitrate concentration as a nitrate transporter.

Keywords. cassava; MeNRT2.1 gene; expression pattern; high-affinity nitrate transporter.

Introduction

Nitrogen is considered to be one of the most essen-tial macronutrients during plant life, and is involvedin the building block of key biological macromolecules(Dechorgnat et al. 2011). Nitrogen deficiency acceleratesthe senescence of older leaves, from which amino acidsand nitrate (inorganic nitrogen) are remobilized to youngleaves or seeds through phloem, thereby leading to growthretardation and yield losses in some plants (Fan et al. 2009;Hsu and Tsay 2013).

Nitrogen exists in soil as both forms, organic andinorganic. Nitrate, ammonium, and urea represent thepredominant inorganic forms of nitrogen, and are oftensupplied as fertilizers; on the other hand, amino acids, pep-tides and proteins are the major sources of organic nitrogenin the boreal-forest soils (Wang et al. 2012). Although,the plants can directly absorb the two forms of nitrogen,

the nitrate and ammonium are the two most readilyavailable inorganic forms of nitrogen for uptake from soil(Zheng et al. 2013). However, ammonium as the sole nitro-gen form represses the elongation of roots (Lima et al.2010; Rogato et al. 2010) and excess ammonium is consid-ered to be toxic to plants (Chiu et al. 2004). For manyplants, nitrate is the preferred nitrogen source. Besidesbeing a vital plant nutrient, nitrate also acts as a signalmolecule (Walch-Liu et al. 2000, 2005; Alboresi et al.2005; Bouguyon et al. 2016). Nitrate concentrations insoil often vary largely. Plants have evolved several absorp-tion and transport systems to adapt to changing sup-plies to optimize nitrate acquisition and support growth:low-affinity transporter system (LATS) and high-affinitytransporter system (HATS) (Forde 2000; Miller et al.2007), including NRT1/PTR family (NPF), nitrate trans-porter 2 (NRT2), chloride channels (CLCs) and slowanion channel-associated 1 homologues (SLAC1/SLAH)

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(Leran et al. 2014; Xia et al. 2015; Fan et al. 2016). NRT2members are responsible for absorption by root from poorsoil with low nitrate concentration. Therefore, the func-tional investigation and characterization of NRT2 familymembers will be important for future applications in sus-tainable agriculture.NRT2 genes were first isolated and characterized from

Aspergillus nidulans (Johnstone et al. 1990; Unkles et al.1991). Subsequently, NRT2 genes have been found inother species, including barley (Trueman et al. 1996), yeast(Perez et al. 1997), tabacoo (Quesada et al. 1997), Ara-bidospis (Zhuo et al. 1999), rice (Cai et al. 2008) etc. InArabidopsis thaliana, seven NRT2 genes have been cloned(Orsel et al. 2002), and some NRT2 transporters aredependent on nitrate assimilation related gene 2 (NAR2),a partner protein, to form complex for their function(Okamoto et al. 2006; Orsel et al. 2006). Until now, atleast four transporter genes have been proposed to betranscriptionally induced in the presence of low nitrateconcentration (<1 mM) and participated in the phase ofnitrate absorption from soil into root (Filleur and Daniel-Vedele 1999; Lejay et al. 1999, 2003, 2008; Zhuo et al.1999; Cerezo et al. 2001; Filleur et al. 2001; Nazoa et al.2003; Okamoto et al. 2003; Munos et al. 2004; Li et al.2007; Tsay et al. 2007; Kiba et al. 2012). In most envi-ronmental conditions, the high-affinity nitrate capacity isprimarily dependent on the AtNRT2.1 protein. HATSactivity is reduced by 63%–75% in the atnrt2 mutant(Cerezo et al. 2001; Filleur et al. 2001; Orsel et al. 2004;Li et al. 2007; Laugier et al. 2012), providing strong evi-dence that NRT2.1 gene is crucially participated in nitratetransport with low concentration. In barley, seven to tenmembers of the NRT2 gene family were found in genomedatabases, of which four were isolated from roots (True-man et al. 1996; Vidmar et al. 2000) and showed nitrateinducible expression. In rice, five NRT2 genes have beenidentified (Cai et al. 2008; Feng et al. 2011; Yan et al.2011; Tang et al. 2012). Besides differences in 5′- and3′- UTRs, an identical coding region has been found inOsNRT2.1 and OsNRT2.2 sequences (Tang et al. 2012).OsNRT2.1 overexpressed in rice slightly improved riceseeding growth, but nitrate absorption was not affected,probably due to lack of coregulation with OsNAR2.1,which is an essential cofactor for high-affinity nitratetransport (Feng et al. 2011; Yan et al. 2011). Recently,transgenic plants expressing the OsNRT2.1, which wasdriven by the nitrate-inducible promoter of the OsNAR2.1gene, exhibited greatly increased biomass improving yieldsby ∼38% and increasing agricultural nitrogen-use effi-ciency (ANUE) to 128% of that of the wild-type plants(Chen et al. 2016). The above evidences prove the impor-tant role of NRT2 family members, especially NRT2.1, inthe efficient absorption of low nitrate concentration andthe increase of biomass.

As the fifth biggest food plant, cassava (Manihot escu-lenta Crantz), belonging to the member of Euphorbiaceae

family is tolerant to drought and low-nutrient soils(Jorgensen et al. 2005; Bredeson et al. 2016). Therefore,cassava is the principal food of many developing countriesin tropic and subtropic areas. However, due to its outcross-ing nature and broad tropical distribution, cassava exhibitshigh heterozygousity (Fregene et al. 2003; Siqueira et al.2010). It is difficult and ineffective to identify poten-tially desirable genes from cassava through conventionalmethods.

Cassava grows well in low-nutrient soils because of itshigh nitrate-absorption activity. Low nitrogen in soil isadvantageous to cassava root differentiation, tuber elonga-tion and augmentation. To understand the molecular basisfor tolerance of poor soil and to improve the utilizationrate of nitrate in cassava, a high-affinity nitrate transportergene which was highly homologous to other NRT2 genes,named MeNRT2.1, has been isolated from root of cassavaseeding (Hu et al. 2016). Compared to other crops, cassavagenetic transformation were greatly delayed, low efficiencyand long cycle from callus culture to seeding emergence.To verify the function as soon as possible, MeNRT2.1 wastransferred into Arabidopsis. Here, we have characterizedthe spatial-temporal expression pattern of the MeNRT2.1through the expression of β-glucuronidase (GUS) reportergene under the control of the native promoter in roots,stems and leaves, in response to different nitrate concen-trations. Further, we have revealed that MeNRT2.1 playsa crucial role in nitrate absorption by the root at extremelylow concentration (0.2 mM NO−

3 ) in transgenic Arabidop-sis plants.

Materials and methods

Construction of proMeNRT2.1::GUS and overexpressing vectors

Approximately 1.9-kb upstream of the inferred ATG startcodon of the MeNRT2.1 gene (cassava4.1_031095m) wasgenerated by polymerase chain reaction (PCR) from cas-sava genomic DNA using PrimeSTAR@Max DNA Poly-merase (Takara, Japan) and gene-specific primers (Pf: 5′-CGCGGATCCATTACTCCCCCTCCATAAGA-3′ andPr: 5′-GGAAGATCTACCATAATGTTTGCTCTCTTCCTGT-3′). The fragments were cloned into pMD18-T(Takara), sequenced, and assembled in front of the GUScoding sequence in the pCAMBIA1301 vector to generateproMeNRT2.1::GUS. To construct MeNRT2.1 overex-pressing plants, full-lengthMeNRT2.1open reading frame(ORF) was amplified from RNA that was extracted fromroots of cassava seedings grown on 0.2 mM NO−

3 MSmedia with special primers: Ff (5′-GGAAGATCTGATGGCTGATATTGAGGGTTCTC-3′) and Fr (5′-GGACTAGTGACATGGACTGGTGTAGTGTTCG-3′). PCR seq-uences were confirmed correct and then inserted down-stream of the 35S promoter in pCAMBIA1302 by BglII and Spe I to produce the plasmid 35S::MeNRT2.1.

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These recombinants were transformed intoAgrobacteriumtumefaciens strain GV4404 by freeze-thawing with liq-uid nitrogen as described previously (Chilton et al. 1974).Transformants were screened on yeast extract and beefplates (YEP) containing rifampicin (100µg/L),chloromycetin (25µg/L) and kanamycin (50µg/L) andverified positive via PCR with primers (Ff and Fr).

Plant transformation and physiological-index measurement

The Arabidopsis wild-type Columbia (Col) strain wastransformed with 35S::MeNRT2.1 constructs by dip-ping their floral buds into an Agrobacterium solution(Clough and Bent 1998). Primary transformants grown at22◦C under continuous light conditions on MS medium(Murashige and Skoog) containing 30 mg/L hygromycin.After two to three generations of selfing, homozygoustransgenic plants were obtained for further study.

Seeds harvested from transgenic lines homozygous for35S::MeNRT2.1 construct were divided into three parts.One was grown on MS medium for 36 days and the othertwo were grown on MS medium for 15 days and furtherone of them was transferred to MS medium contain-ing 2 mM KClO3 for three days (Tsay et al. 1993), andthe other was treated with 0.2 mM NO−

3 MS medium for∼15 days. Phenotypes of transgenic plants after treat-ment were photographed. Fresh weight and root lengthwere determined and content of nitrate reductase wasmeasured according to the kit instructions (Suzhou Com-inbio Biological Technology). Total chlorophyll from freshleaves was calculated as described (Lichtenthaler 1987;Bagchi et al. 2012).

Histochemical observation of GUS

Seeds of transgenic lines homozygous for the proMeNRT2.1::GUS construct were selected from T3 transformantswhich grown on MS medium for three weeks. Then allthe seedlings were respectively transferred to 0 mM nitrate,0.2 mM nitrate, or full-N (25 mM nitrate and 2.5 mMammonium succinate) and free-N (N starvation) MSmedium for treatment for 1, 5, 12, 24 and 72 h. Hand-cutsections of developing roots, stems and leaves were stainedwith buffer for 24 h at 37◦C and were then rinsed with agraded ethanol series (Lee et al. 2009). The images werephotographed under stereoscope (Leica).

Quantitative real-time PCR

Total RNA of wild-type and overexpressing plants wasextracted using TRIzol (Invitrogen). The first strandcDNA of each sample was synthesized according to thePrimeScript RT Enzyme kit (TaKaRa) manufacturer’sinstructions. qRT-PCR was performed on Applied Biosys-tems StepOnePlus RT-PCR System Thermal Block (Life

technologies) with SYBR Green mix. The primers forMeNRT2.1 were Qf (5′-CTCTAGGAGGTTCCTTCTGCATCT-3′) and Qr (5′-TTTCCACCAGCACCAGTCAATC-3′). The procedure of PCR amplification was as fol-lows: a 30 s denaturing step at 95◦C, 5 s at 95◦C and30 s at 60◦C with 45 cycles. qRT-PCR of each sam-ple was performed thrice with biological significance,and threshold cycle values were quantified by qRT-PCRaccording to the previous report (Livak and Schmittgen2001). The Arabidopsis ACTIN gene was selected asan internal standard to normalize the expression ofMeNRT2.1 (sequences of primer for amplification were 5′-TGAGAGATTCCGTTGCCCAGAAGT-3′ and 5′- TTCCTTACTCATGCGGTCTGCGAT-3′).

15NO−3 measurement in overexpressing plants

After germination of wild-type and overexpressing seedsgrown on 1/4 MS medium, all seedlings of consistentgrowth were shifted to full-N MS for three weeks, andthen transferred to hydroponic solution and cultivated fortwo weeks with the environmental parameters as describedpreviously (Lin et al. 2008). The nutrient solution waschanged twice a week during the first week of cultureand was changed every day in last week. Shoots were sup-ported on 1–2 cm holes of foam board with all the rootsimmersed in the culture solution. Nitrate uptake using15NO−

3 was determined according to previous references(Remans et al. 2006; Laugier et al. 2012). The plants werewashed with 0.1 mM CaSO4 for 1 min, and then trans-ferred for 30 min to fresh nutrient medium containingK15NO3 with a 99% atom excess of 15N at the concentra-tions indicated in the figures, and finally washed thrice in0.1 mM CaSO4. Total 15N content was measured using acontinuous-flow isotope ratio mass spectrometer coupledwith a carbon nitrogen elemental analyser (ANCA-MS;PDZ Europa). The values are means of six replicates.

Results

MeNRT2.1 is induced by low nitrate concentrations in roots,leaves and stems

To further investigate the spatiotemporal expression pat-tern of MeNRT2.1, we fused the GUS reporter gene to thepromoter region of the MeNRT2.1 gene (proMeNRT2.1::GUS construct) and introduced this construct into Ara-bidopsis plants via the floral dip method. The homozygouslines for theproMeNRT2.1::GUSwere selected, and grownon MS medium for three weeks. Then, all the seedlingswere transferred to 0 mM nitrate, 0.2 mM nitrate, or full-N(nitrate and ammonium succinate) and free-N (N star-vation) MS medium for treatment for 1, 5, 12, 24 and72 h. Six independent transgenic plants were stained withGUS activity for each treatment and then photographed

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Figure 1. GUS staining in different tissues of seedlings grown on MS media containing various concentrations of KNO3 and (NH4)2succinate. (a) GUS expression of seedlings grown under full N conditions is only found in roots in examination times. (b) GUS signalsare detectable in roots at all the indicated times, but leaves and stems show strong staining at 12 h during no nitrate but (NH4)2succinate. (c) Slightly GUS staining is seen in roots at all the indicated times, but leaves indicate strong GUS signals at 12 h duringN starvation. (d) Slightly GUS activity in roots is found after 1 h, stronger staining are seen after 5 h, the highest strong expressionis detected after 12 h under 0.2 mM nitrate condition. The staining of leaves and stems is observed only at 12 h.

for each tissue. The GUS gene under the promoter ofthe MeNRT2.1 gene slightly showed expression in root ofseedlings grown under full-N condition in all examinationtimes (figure 1a). After treatment with MS medium, withno nitrate but ammonium succinate, GUS signals weredetected in roots, at all indicated time, while leaves andstems showed strong staining at 12 h (figure 1b). Theseresults were similar to the free-N condition except thatGUS activity was absent in stem (N starvation, figure 1c).Modest GUS activity in roots was observed after 1 h, withstronger staining after 5 h and strongest expression after12 h under 0.2 mM nitrate conditions. However, stainingin leaves and stems was restricted to 12 h (figure 1d). Theseresults indicated that the MeNRT2.1 gene is basicallyexpressed in roots, but is induced by low nitrate concen-tration in roots, leaves and stems (0.2 mM NO−

3 ).

Overexpression of MeNRT2.1 sensitizes Arabidobsis to chlorate

As an herbicide and defoliant, chlorate is a nitrate analogthat is taken up by nitrate transporters and then reducedby nitrate reductase to chlorite, which is toxic to plants(Tsay et al. 1993). Therefore, chlorate has been used toscreen for mutants defective in nitrate uptake or reduction

(Liu et al. 1999). As shown in figure 2, when plants weretreated with 2-mM chlorate in MS medium, severe chloro-sis was observed in overexpressing Arabidopsis plantscarrying 35S::MeNRT2.1 which were able to ingest morechlorate, as well as nitrate, but the wild-type plants weremore resistant under the same conditions (figure 2a). Inter-estingly, when the overexpression and wild-type plantswere shifted to normal MS medium for 24 h after chlo-rate treatment, neither of them showed signs of chlorosis(figure 2b).

Enhanced 15NO−3 uptake activity of overexpressing plant in low

nitrate concentration

To directly measure the nitrate uptake, lines harbouring35S::MeNRT2.1 were grown on N-full MS medium forthree weeks and then transferred into 15NO−

3 medium for30 min. The 35S::MeNRT2.1 lines had no obvious phe-notypes when grown on the N-full MS medium in theearly growth stage. However, after 36 days, leaf numberin 35S::MeNRT2.1 lines (10 ± 2) was more than in wild-type plants (6 ± 1) (figure 3a). In addition, the fresh weightand root length of overexpressing lines (fresh weight:100.3 ± 23.5 mg/line; root length: 73.4 ± 2 mm/line) wassignificantly increased over wild-type plants (fresh weight:

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Figure 2. Morphology of Arabidopsis plants treated with chlo-rate. (a) Wild type (WT) and overexpression (OE) 15-day-oldplants carrying the cassava 35S::MeNRT2.1 construct weretreated with 2 mM KClO3 three days. (b) Photographs were takenof WT and OE plants grown 24 h on normal MS media aftertreatment with chlorate.

51.2 ± 1.2 mg/line; root length: 52.1 ± 6 mm/line) (figure 3,a, d & f). Notably, chlorophyll content in 35S::MeNRT2.1lines was also higher than wild-type plants (figure 3e),suggesting that MeNRT2.1 could promote growth anddevelopment of Arabidopsis on N-full medium consis-tent with the results of GUS assay (figure 1a). However,althoughMeNRT2.1mRNA levels were dramatically highin 35S::MeNRT2.1 lines (figure 3b), 15NO−

3 uptake andnitrate reductase content were only modestly higher in35S::MeNRT2.1 plants (figure 3, c&g). This suggests thatthere is a strong post-transcriptional regulation betweenMeNRT2.1 mRNA expression level and root NO−

3 influx,in agreement with regulation of NRT2.1 in other systems(Fraisier et al. 2000; Laugier et al. 2012).

Because MeNRT2.1 is probably a high-affinity trans-porter, NO−

3 root uptake of 35S::MeNRT2.1 plants ismore interesting under low nitrate conditions. Seedlingsgrown on normal MS medium for 15 days were shiftedto 0.2 Mm NO−

3 MS medium containing for 15 dayswhere wild type became yellow and even died, while35S::MeNRT2.1 plants showed normal growth except 2–3yellow leaves at the bottom of the plant (figure 4a). Therewere no differences in fresh weight or root length (figure 4,d&f). However, chlorophyll content, 15NO−

3 absorption

Figure 3. Characteristic of overexpression plants grown withN-full on MS media. (a), (d) and (f), Morphological phenotypes(a) of wild type (WT) and overexpression (OE) plants carrying35S::MeNRT2.1 construct show that the fresh weights of over-expression lines was higher than those of wild type (d), the rootsof overexpression lines were longer than those of wild type (f),red bar = 2 cm. (b) qRT-PCR analysis of MeNRT2.1 expressionshows that the overexpression lines (OE) had higher expressionlevel than the wild type (WT). (c), Root NO3 uptake in the wildtype (WT) and overexpression lines (OE). DW, dry weight. (e)and (g), Chlorophyll and nitrate reductase contents of wild type(WT) and overexpression plants (OE). Above data show meansand SD values of biological replicates (n > 3) and statistical anal-ysis by heteroscedastic t-test indicating differences (*P < 0.5) orsignificant differences (**P < 0.01).

and nitrate reductase content were all dramatically higherin 35S::MeNRT2.1 than in wild-type plants (figure 4, b, c,e&g), these results indicate that MeNRT2.1 function as ahigh-affinity transporter.

Discussion

The importance of high-affinity nitrate transporters innitrogen use efficiency has been previously demonstratedand been appreciated (Okamoto et al. 2003; Orsel et al.2004; Feng et al. 2011; Kiba et al. 2012; Tang et al. 2012;Chen et al. 2016). RNA-seq technology was used to isolatea putativeNRT2.1 (MeNRT2.1) from cassava in our previ-ous research (Hu et al. 2016).MeNRT2.1 encodes an ORFof 1593 bp corresponding to a protein of 530 amino acidresidues, which display 90% identity to the Theobromacacao NRT2.1. MeNRT2.1 has classical topology consistsof 12 putative transmembrane segments, is localized on

74 Page 6 of 9 Liangping Zou et al.

Figure 4. Characteristic of overexpression lines grown with0.2 mM nitrate on MS media. (a), (d) and (f). Morphologicalphenotypes of wild type (WT) and overexpression (OE) plantscarrying 35S::MeNRT2.1 construct show that all yellow leaves ofwild type were observed after treatment with 15 days on 0.2 mMnitrate media, while most of leaves of overexpression lines stillwere green under the same condition (a). But the fresh weights(d) and root length (f) did not differ between wild-type andoverexpression lines. Red bar = 2 cm. (b) qRT-PCR analysis ofMeNRT2.1 expression shows that the overexpression lines (OE)had higher expression level than the wild type (WT). (c) RootNO3 uptake in the wild type (WT) were significant lower thanoverexpression lines (OE). DW, dry weight. (e) and (g), chloro-phyll and nitrate reductase contents of wild type (WT) were moresignificant decrease than overexpression plants (OE). Above datashow means and SD values of biological replicates (n > 3) andstatistical analysis by heteroscedastic t-test indicating significantdifferences (**P < 0.01).

the plasma membrane and gets induced mainly in root by0.2 mM nitrate (Hu et al. 2016).

Intensive spatiotemporal expression profiling ofNRT2.1has been established, and the molecular mechanism ofthis high-affinity nitrate activity in Arabidopsis has alsobeen illuminated (Okamoto et al. 2003; Kiba et al. 2012).However, it is obscure whether the same expressionpattern of NRT2.1 holds in cassava, which grows wellon low-nutrient soils. To gain insight into the function ofcassava NRT2.1, transgenic Arabidopsis plants of hetero-geneously expressional ProMeNRT2.1::GUS were usedto investigate its expression pattern. GUS reporter analy-ses revealed that MeNRT2.1 was constitutively expressedin roots (figure 1, a,b&c), implying that MeNRT2.1 hasa certain basal expression that differs from NRT2.1 inArabidopsis, which is transiently increased by N starva-tion (Krapp et al. 2014). However, induced expression wasfound in roots after 12 h under 0.2 mM nitrate conditions

(figure 1d). The different NRT2.1 expression betweenArabidopsis and cassava suggested a evolutionary diver-gence in its regulation. In addition, MeNRT2.1 expressionwas induced in leaves after treatment for 12 h under N-freeconditions (figure 1b), suggesting a unique high-affinityscavenging role for cassava NRT2.1 in NO−

3 remobilizationduring N starvation, just like NRT2.4 or NRT1.7 inArabidopsis (Fan et al. 2009; Kiba et al. 2012). Cas-sava, the major cultivated species of the genus Manihot,is well adapted to poor soil and high yielding, whichsuggests that it has highly efficient system for using nutri-ents in soil. To avoid unnecessary waste, it is importantto finely adjust gene expression to achieve maximumefficiency for one gene through different spatiotempo-ral expression pattern. Therefore, MeNRT2.1 was alsoexpressed in stem and leaf along with induced expres-sion in roots after treatment with 0.2 mM NO−

3 for 12 h(figure 1d).

Transcriptional and post-transcriptional regulation mayprovide a fine-tuning for functions of nitrate transportergenes. Nitrate, nitrite, ammonium, glutamine, N starva-tion, light, sucrose, diurnal rhythm, and/or pH can regulateexpression of CHL1 (AtNRT1.1), AtNRT1.2, AtNRT2.1,AtNRT2.2, and/or AtNRT2.4 at the transcriptional level(Wang et al. 2012). Studies of transgenic Arabidopsisexpressing a functional 35S::NRT2.1 have revealed thatpost-transcriptional regulation plays a predominant rolein the control of the NO−

3 HATS. HATS-mediated 15NO−3

influx was reduced in overexpressing lines at 0.2 mM or10 mM NO−

3 (Laugier et al. 2012). However, in this study,15NO−

3 uptake was not increased in overexpressing plantsgrown on N-full medium but was dramatically increasedin overexpressing lines at 0.2 mM NO−

3 (figures 3c; 4c),suggesting that the post-transcriptional regulation ofMeNRT2.1 gene has been particularly linked to responseto different nitrate concentration. High NO−

3 uptakeinduces corresponding nitrate reductase gene expressionwhich results in increased nitrate reductase content inoverexpressing plants in the presence of 0.2 mM NO−

3 (fig-ure 4g). In addition, in hydroponic cultures with nitrate asthe sole N source, the overexpressing plants displayed sig-nificantly increased chlorophyll content, and senescencewas delayed increasing survival and preventing emergenceof yellow leaves under extremely low nitrate conditions(figures 3a & 4a).

Interestingly, 35S::MeNRT2.1 transgenic lines exhib-ited dramatically increased fresh weight and root lengthunder N-full condition but not under 0.2 mM NO−

3 con-ditions (figures 3, d&f; 4, d&f). Perhaps nitrate, as asignalling molecule, participates in regulating root elon-gation in nitrate-rich patches of the external medium(Krouk et al. 2010), which promotes growth and therebyincreases biomass of transgenic plants (Chen et al. 2016).However, higher NO−

3 uptake in overexpressing plantsonly meets the requirement to maintain viability undersuch low concentrations of nitrate.

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Specific functional studies of MeNRT2.1 in cassavavial loss-of-function and gain-of-function mutants is stillneed to be performed. Further, some high-affinity nitratetransporters belonging to the NRT2 family have beenshown to depend on expression of the NAR2-like genefor their function (Okamoto et al. 2006; Orsel et al.2006; Feng et al. 2011). It will be important to determinewhether MeNRT2.1 also requires a NAR2 partner proteinin cassava.

Acknowledgements

This work was supported by the National Key R&D Programmeof China (grant no. 2018YFD1000500), the national key technol-ogy R&D programme of China (grant no. 2015BAD15B01) andawards for excellent researcher from Chinese Academy of Tropi-cal Agricultural Sciences (to M.P). Thank David Pincus workedin Whitehead Institute for Biomedical Research, Cambridge forrevision in all my paper.

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Corresponding editor: Umesh Varshney