REVIEW

Improving coordination of plant growth and nitrogenmetabolism for sustainable agriculture

Xiang Han1 , Kun Wu1 , Xiangdong Fu1,2 , Qian Liu1&

1 The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and DevelopmentalBiology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China

2 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

Received: 17 April 2020 / Accepted: 20 July 2020 / Published online: 31 August 2020

Abstract The agricultural green revolution of the 1960s boosted cereal crop yield was in part due to cultivationof semi-dwarf green revolution varieties. The semi-dwarf plants resist lodging and require high nitrogen(N) fertilizer inputs to maximize yield. To produce higher grain yield, inorganic fertilizer has beenoverused by Chinese farmers in intensive crop production. With the ongoing increase in the fooddemand of global population and the environmental pollution, improving crop productivity withreduced N supply is a pressing challenge. Despite a great deal of research efforts, to date only a fewgenes that improve N use efficiency (NUE) have been identified. The molecular mechanisms underlyingthe coordination of plant growth, carbon (C) and N assimilation is still not fully understood, thuspreventing significant improvement. Recent advances have shed light on how explore NUE within anoverall plant biology system that considered the co-regulation of plant growth, C and N metabolisms asa whole, rather than focusing specifically on N uptake and assimilation. There are several potentialapproaches to improve NUE discussed in this review. Increasing knowledge of how plants sense andrespond to changes in N availability, as well as identifying new targets for breeding strategies tosimultaneously improve NUE and grain yield, could usher in a new green revolution.

Keywords Green revolution, Yield, Nitrogen use efficiency, Growth-metabolism coordination, Breeding strategy

INTRODUCTION

N nutrients are essential for sustaining plant growthand development, the availability of N in the soil is amajor limiting factor for crop yield. In the 1960s, thegreen revolution enhanced cereal crop yield, fed agrowing human population, and was in part due towidespread adoption of semi-dwarf varieties (Khush1999). The beneficial semi-dwarfism is conferred by themutant alleles at wheat Reduced height-1 (Rht-1) andrice semi-dwarf1 (sd1) loci, respectively (Peng et al.1999; Sasaki et al. 2002). Under high N conditions,green revolution varieties (GRVs) exhibit a reduced risk

of grain yield loss due to plant flattening by wind andrain (the yield-reducing phenomenon known as ‘lodg-ing’). Both sd1 and Rht-1 alleles are still widely usednow to produce new elite varieties of rice and wheat.Normally, the phytohormone gibberellin (GA) promotesplant growth by destructing the growth-repressingDELLA proteins (Sasaki et al. 2003; Fu et al. 2004). Thewheat mutant Rht-1 protein likely resists GA-stimulateddestruction (Peng et al. 1999), while the rice mutant sd1allele reduces GA abundance and consequently increa-ses accumulation of the rice DELLA protein SLR1 (Liet al. 2018). It has been shown that GRVs have a rela-tively poor NUE, require a high N fertilizer input toachieve maximum yield potential (Gooding et al. 2012;Li et al. 2018). To increase cereal crop yields, excessive

& Correspondence: liuqian0726@163.com (Q. Liu)

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chemical N fertilizer has been used by famers. There-fore, a major challenge for sustainable agriculture iswhether improvement of NUE through the reduction inthe amount of N fertilizer can be achieved without thecorresponding yield penalty.

NUE is a measure of plant ability to use soil N (in-cluding the residual N nutrients present in the soilsystem and those provided by fertilization). Given theuncertainty of residual N in the soil, we usually simplydefine cereal crop NUE as grain yield per unit of sup-plied fertilizer (Moll et al. 1982; Han et al. 2015). NUE incrop plants is a complex quantitative trait, which iscontrolled by quantitative trait loci (QTL) and influ-enced by multiple environmental factors. In the pastdecade, a lot of transporting and metabolism genes thatprovide better access to the biological processes of Nuptake, transport, assimilation and redistribution havebeen identified in model plants (Li et al. 2017; Wanget al. 2018d; Vidal et al. 2020). In addition, a number ofN-responsive genes and relevant regulatory elements inthe control of N uptake and plant architecture inresponse to changes in N availability have also beeninvestigated (Li et al. 2017; Wang et al. 2018d; Jia andWiren 2020; Luo et al. 2020). Although many effortshave been focused how to improve grain yield and NUE,the molecular mechanisms underlying N sensing andsignaling networks are still elusive, and the NUEimprovements in crops have a limited success. Recently,several genes that improve rice GRVs’ NUE have beenreported, the products of which have been demon-strated to be involved not only in regulating N assimi-lation, but also in improving coordination of plantgrowth, C and N metabolism (Li et al. 2018; Wu et al.2020b). In this review, we summarize some recentadvances in understanding of the molecular mecha-nisms underlying NUE improvements and co-regulationof C–N balance, and also review some biotechnologicaland breeding strategies through modulation of plantgrowth-metabolism coordination for future sustainablefood security and a new green revolution.

REGULATION OF N ACQUISITION AND ASSIMILATION

As sessile organisms, plant roots are able to acquire awide variety of N forms through transmembranetransporters or channels, ranging from simple inorganicN compounds such as ammonium (NH4

?) and nitrate(NO3

-) to polymeric N forms such as amino acids andother organic N forms. However, plant roots take upmainly inorganic NH4

? and NO3- from the soils, rather

than organic N forms. In most soils, NO3- concentration

can vary dramatically, a high-affinity transport system

(when the external NO3- concentration is low, e.g., \

1 mM) and a low-affinity transport system (when theexternal NO3

- concentration is high, e.g.,[ 1 mM) areinvolved in NO3

- absorption (Miller et al. 2007). Aftertaken up by nitrate transporters (NRTs), part of NO3

-

could be reduced in roots immediately, the rest of NO3-

will be translocated to the shoots for assimilation, andthe proportion of reduction is dependent on the plantspecies and N-supply levels. In contrast, the ammoniumtransporter/methylamine permease/Rhesus (Amt/Mep/Rh) family proteins mainly account for high-affin-ity NH4

? uptake capacity (Khademi et al. 2004; Yuanet al. 2007). In most plants, almost all the absorbedNH4

? is assimilated into glutamine (Gln) locally in theroots rather than being translocated to the shootsbecause of its toxicity (Britto et al. 2001). The trans-porters of NO3

- and NH4? have been identified and

functionally characterized in Arabidopsis and severalcrop species, and the regulatory mechanisms affecting Nuptake have been extensively investigated.

Nitrate transporters

For most plants, NO3- is the main source of soil N

supply. There are four distinct protein families oftransmembrane transporters or channels involved inNO3

- absorption and translocation, including NRT1/PTR (nitrate transporter1/peptide transport family,hereafter referred to as NPF), NRT2, CLC (chloridechannels), and SLAC (slow-type anion channel)/SLAH(slow anion channel-associated homologs). NPF is thebiggest family that have 53 members in Arabidopsis,most of which function as low-affinity transporters.However, there are three exceptions to this functionalseparation, Arabidopsis AtNRT1.1/AtNPF6.3, riceOsNPF2.4, and Medicago MtNRT1.3, which have beenreported to display dual affinity for NO3

- (Liu et al.1999; Morere-Le Paven et al. 2011; Wei et al. 2018). Ithas been shown that AtNRT1.1/AtNPF6.3 andAtNRT1.2/AtNPF4.6 are predominantly involved inNO3

- uptake in the roots (Huang et al. 1996, 1999),AtNRT1.7/AtNPF2.13, AtNRT1.11/AtNPF1.2 andAtNRT1.12/AtNPF1.1 mainly mediate NO3

- redistribu-tion from old leaves to young tissues (Fan et al. 2009;Hsu and Tsay 2013), and other NPF transporters, suchas AtNRT1.5/AtNPF7.3, AtNRT1.8/AtNPF7.2, AtNPF2.3,AtNRT1.9/AtNPF2.9 play key roles in facilitating long-distance NO3

- translocation (Lin et al. 2008; Li et al.2010; Wang and Tsay 2011; Taochy et al. 2015). Inaddition, NPF family members have been shown totransport various other substrates, including GA, auxin,abscisic acid (ABA), jasmonoyl-iso-leucine, and

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glucosinolates (Krouk et al. 2010a; Nour-Eldin et al.2012; Chiba et al. 2015).

In rice, there exists 93 homologs of the NPF familytransporters, most of which have not yet been func-tionally characterized (Wang et al. 2018d). OsNRT1/OsNPF8.9 was the first identified transporter involvedlow-affinity uptake of NO3

- (Lin et al. 2000), the up-regulation of OsNRT1/OsNPF8.9 promotes NO3

- uptakeand rice growth (Fan et al. 2016a). OsNRT1.1B/OsNPF6.5 is a rice sequence homolog of AtNRT1.1/AtNPF6.3. The osnrt1.1b mutant exhibits reduced NO3

-

uptake and root-to-shoot translocation, whereas higherexpression of OsNRT1.1B causes increases in N uptake,biomass production and grain yield (Hu et al. 2015).OsNRT1.1A/OsNPF6.3, another sequence homolog ofAtNRT1.1/AtNPF6.3, is predominantly localized to thetonoplast, which has also been shown to improve NUEand rice yield (Wang et al. 2018c). The rice mutantslacking OsNPF2.2 exhibit impaired NO3

- translocationfrom roots to shoots, unloading from the xylem (Li et al.2015). OsNPF2.4 is shown to been an all-rounder,because it functions in almost all processes, includingNO3

- uptake, root-to-shoot translocation and source-to-sink redistribution (Xia et al. 2015). OsNPF7.1,OsNPF7.2, OsNPF7.3, OsNPF7.4, and OsNPF8.20/OsPTR9 are shown to promote N assimilation, tillerbranching and rice yield (Fang et al. 2013, 2017; Huet al. 2016; Wang et al. 2018a; Huang et al. 2019). Inaddition, neither NO3

- nor NH4? transporter activity of

OsNPF4.1/SP1 (short panicle1) has been detectedin vivo and in vitro (Li et al. 2009b). Recently, Genome-wide association studies (GWAS) of NUE-related agro-nomic traits in rice collection have identified two newgenes, OsNPF6.1 and OsNAC42, which are associatedwith increased rates of NO3

- uptake (Tang et al. 2019).Interestingly, a rare allele of OsNPF6.1 derived fromthe variation in wild rice, which allele-specificexpression of OsNPF6.1 is much more likely to betransactivated by OsNAC42, confers increased NUEand rice yield.

High-affinity NRT2 family transporters compriseseven genes in Arabidopsis (Orsel et al. 2002). AtNRT2.1and AtNRT2.2 are found to play a major role in NO3

-

uptake from soil, whereas AtNRT2.4 and AtNRT2.5especially function in young roots under low N condi-tions (Kiba et al. 2012; Lezhneva et al. 2014). Theactivities of AtNRT2 family transporters require a criti-cal partner protein AtNAR2.1 (nitrate assimilationrelated protein, also known as AtNRT3.1), and theAtNRT2–AtNAR2.1 interaction can stabilize AtNRT2protein and its plasma membrane localization (Koturet al. 2012). It is found that AtNRT2.7 does not interactdirectly with AtNAR2.1, and the tonoplast-localized

AtNRT2.7 is shown to mediate NO3- accumulation in

vacuoles (Chopin et al. 2007; Kotur et al. 2012).In rice, there are four OsNRT2 and two OsNAR2 genes

(Orsel et al. 2002; Cai et al. 2008). The OsNRT2.3 genecan be mRNA-spliced into OsNRT2.3a and OsNRT2.3bisoforms. OsNRT2.3a is mainly involved in long-distanceNO3

- transport from roots to shoots (Tang et al. 2012),whereas OsNRT2.3b acts as a sensor to switch NO3

-

transport activity on or off by a pH-sensing mechanism(Fan et al. 2016b). Importantly, the up-regulation ofOsNRT2.3b enhances the pH-buffering capacity of therice plants in response to varied N supply, consequentlyresulting in the significant improvements of NUE andgrain yield in rice (Fan et al. 2016b). The functionaltransport activities of OsNRT2.1, OsNRT2.2 andOsNRT2.3a depend on their interactions with OsNAR2.1(Yan et al. 2011), overexpression of either OsNRT2.1 orOsNAR2.1 enhances NO3

- uptake and grain yield (Chenet al. 2017; Luo et al. 2018). In contrast, OsNRT2.3b andOsNRT2.4 function independently to regulate shootdevelopment and lateral root growth without a partnerprotein OsNAR2 (Wei et al. 2018). Unlike Arabidopsis,diversity analysis revealed a significant genetic distanceamong the NRT2 gene family in cereal crops (Buchnerand Hawkesford 2014), implying that proper functionalanalysis in cereal crops need to be investigated forverifying NRT2 function.

Ammonium transporters

NH4? is the main source of soil N supply for plants

grown in flooded wetland or acidic soils. The Amt/Mep/Rh family proteins have been demonstrated to facilitatethe movement of NH4

? cross the membrane. In Ara-bidopsis, there exists six AMT-type NH4

? transporters.Thereinto, AtAMT1;1 (At4g13510), AtAMT1;2(At1g64780), AtAMT1;3 (At3g24300), AtAMT1;4(At4g28700) and AtAMT1;5 (At3g24290) belong toAMT1 subclass, while AtAMT2;1 (At2g38290) is moreclosely related to bacterial AmtB and yeast MEP pro-teins (Ludewig et al. 2007). With the exception ofAtAMT1;4, the AtAMT genes are highly expressed inArabidopsis roots (Gazzarrini et al. 1999). AtAMT1;1and AtAMT1;3 are localized within the root epidermaland cortical cells (Kaiser et al. 2002; Loque et al. 2006),while AtAMT1;2 is localized in the plasma membrane ofthe root endodermis, and plays important roles not onlyin NH4

? uptake and retrieval from root apoplast, butalso in NH4

? translocation to the vasculature (Neu-hauser et al. 2007; Yuan et al. 2007). NH4

? uptakecapacity in the atamt1;1 atamt1;2 atamt1;3 atamt2;1quadruple mutant is reduced by 95% compared withwild-type plants (Yuan et al. 2007), suggesting that

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AtAMTs function predominantly to mediate NH4?

uptake in Arabidopsis.In rice, twelve putative rice OsAMT transportes are

identified based on amino acid sequence homology withAtAMTs, and further divided into five subfamilies,OsAMT1 to OsAMT5 (Li et al. 2009a). The OsAMT1subfamily consists of three distinct members, OsAMT1;1(Os04g0509600), OsAMT1;2 (Os02g0620600) andOsAMT1;3 (Os02g0620500). OsAMT1;1 is constantlyexpressed in both roots and shoots, whereas OsAMT1;2and OsAMT1;3 exhibit the root-specific expression pat-terns (Sonoda et al. 2003). In contrast to NH4

?-inducedexpressions of both OsAMT1;1 and OsAMT1;2, OsAMT1;3is induced by N deprivation, and predominantlyexpressed in root apices, suggesting that OsAMT1;3 mayfunction as an NH4

? sensor (Yao et al. 2008; Ferreiraet al. 2015). Overexpression of OsAMT1;1 significantlyenhances NH4

? uptake in the roots, indicating thatOsAMT1;1 has the potential for improving NUE (Rana-thunge et al. 2014). However, overexpression ofOsAMT1;1 has also been reported to inhibit plantgrowth (Hoque et al. 2006). To date, many efforts toimprove NUE in crops through modulations of the AMTgenes’ expressions have had only limited success, pos-sibly indicating toxicity effects of an excessive NH4

? inplants (Ranathunge et al. 2014; Bao et al. 2015a; Li et al.2016).

NITRATE REDUCTION AND AMMONIUMASSIMILATION

Once NO3- is incorporated into root cells, it is either

reduced or stored in the vacuoles or is translocated tothe shoot for subsequent assimilation and vacuolarstorage. Normally, NO3

- is reduced to nitrite by nitratereductase (NR, also known as NIA) in the cytosol, andthen transferred to the plastid and chloroplast, where itis further reduced to NH4

? by nitrite reductase (NiR)(Liu et al. 2015). There exists two AtNIA genes in Ara-bidopsis and three OsNIA genes in rice, respectively(Cheng et al. 1988; Choi et al. 1989; Wilkinson andCrawford 1991). Compared to wild-type plant, the at-nia2 mutant exhibits * 90% reduced NR activity, andthe atnia1 atnia2 double mutant has * 0.5% NRactivity (Wilkinson and Crawford 1993), indicating thatAtNIA2 plays an important role in regulating NO3

-

reduction.Chlorate, as a transport analog for NO3

-, is taken upby roots and further reduced to toxic chlorite by NR inshoots. A rice NR-deficient mutant M819 that exhi-bits * 90% reduced leaf NADH-NR activity was iden-tified by screening chlorate resistance (Hasegawa et al.

1992). It has long been known that chlorate resistanceis one of the most reliable differentiation between indicaand japonica rice verities. A new finding revealed thatthe allelic variation at OsNR2 locus, the gene encoding aNADH/NADPH NR, is account for the occurrence of thisdifferentiation. The elite indica OsNR2 allele confersincreases in NR activity, tiller number and NO3

- uptakerate via a positive feed-forward regulation on theexpression of OsNRT1.1B, consequently resulting inimproved NUE and grain yield (Gao et al. 2019b).Besides that, the indica varieties are not easy to cultureand regenerate plants compared to japonica rice vari-eties. By conventional crosses of low-regeneration ricecultivar Koshihikari with high-regeneration rice cultivarKasalath, a quantitative trait locus qPSR1, the geneencoding a ferredoxin-NiR (Fd-NiR), has been shown tocontrol the regeneration ability, suggesting that NO3

-

metabolism play an important role in rice regeneration(Nishimura et al. 2005).

Because of toxicity problems, NH4? needs to be

rapidly assimilated and then transported mainly in theform of Gln (Fukumorita and Chino 1982). The glu-tamine synthases (GS) catalyze the fixation of NH4

? on aglutamate (Glu) molecule to produce Gln. Subsequently,Gln reacts with 2-oxoglutarate (2-OG) to produce twomolecules of Glu in a reaction catalyzed by the gluta-mate synthases (GOGAT). There are two type GOGAT,which may use either reduced ferredoxin (Fd-GOGAT)or NADH (NADH-GOGAT) as an electron donor. In rice,there are three genes encoding for cytosolic OsGS1(OsGS1;1, OsGS1;2, and OsGS1;3) and one gene encodingfor chloroplast GS2 (OsGS2). The osgs1;1mutant exhibitssevere reduction in plant growth and abnormal accu-mulation of sugar and organic N forms (Tabuchi et al.2005; Kusano et al. 2011). The osgs1;2 mutant causes areduction in out-growth of tiller buds and a symptom ofC-N metabolic disorder (Ohashi et al. 2015). The osgs1;3mutant exhibits only a reduced rate of natural senes-cence in paddy field (Yamaya and Kusano 2014).Although overexpressions of either or both OsGS1;1 andOsGS1;2 could increase GS activity, the transgenic riceplants exhibit growth retardation, unbalanced C–Nmetabolism and grain yield loss (Cai et al. 2009; Baoet al. 2014). The osgs1;1 mutant displays reduced grainfilling rate, overexpression of either OsGS1;2 or OsGS1;3does not compensate for OsGS1;1 function (Tabuchiet al. 2005; Kusano et al. 2011). Moreover, co-overex-pression of OsGS1;1 and OsGS2 promotes plant growth,photosynthetic and agronomic performance understress conditions (James et al. 2018).

There are two isoforms of NADH-GOGAT (NADH-GOGAT1 and NADH-GOGAT2) in rice. The reversegenetics researches indicated that GS1;2 and NADH-

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GOGAT1 are mainly account for the primary NH4?

assimilation in roots, while OsGS1;1 and OsNADH-GOGAT2 are expressed in vascular tissues of matureleaves, and act as key enzymes during N remobilizationfrom leaves to seeds (Yamaya and Kusano 2014). In therice grains, approximately 80% of total N nutrients arederived from senescent organs through the phloem.Accordingly, OsGS1;1 and OsNADH-GOGAT2 play keyroles in N redistribution and reutilization (Yamaya andKusano 2014). It is worth noting that the osnadh-gogat2mutant is able to grow well during vegetative stage,similar to wild-type plants in the paddy field. But at theharvest time, the osnadh-gogat2 mutant exhibits severereduction in spikelet number and grain yield (Tamuraet al. 2011).

In addition, NH4? is also produced by both pho-

torespiration and amino acid recycling, and in rice, thatis mostly assimilated in leave chloroplasts by OsGS2 andOsFd-GOGAT (Wallsgrove et al. 1987). The OsFd-GOGATnull mutation causes a lethal phenotype (Coschiganoet al. 1998), whereas a weak mutant allele osfd-gogatconfers typical N-deficient syndromes (Yang et al.2016). Loss-of-function mutation of ARE1 gene canrescue the N-deficient syndrome caused by the reducedOsFd-GOGAT activity, and then enhance plant NUE andgrain yield in rice (Wang et al. 2018b). OsGS2-cosup-pressed plants display a normal growth phenotype atthe early seedling stage, while at the tillering stage,transgenic rice plants exhibit a typical N-deficient syn-drome (Cai et al. 2010).

More importantly, up-regulations of some N meta-bolism genes in different crop species, for example,OsGS2 in rice (James et al. 2018), TaGS2-2 in wheat (Huet al. 2018), HvGS1-1 in barley (Gao et al. 2019a), andGln1-3 and Gln1-4 in maize (Martin et al. 2006), havebeen shown to increase N uptake and assimilation, andconsequently enhance plant NUE and grain yields(Fig. 1). However, increased expression levels of these Nmetabolism genes are often associated with increasedplant height, and resultant plants are becoming tallerand more sensitive to lodging than wild-type plants. Theefforts in improving GRVs’ NUE and grain yield withoutloss of the yield-enhancing dwarfism have had limitedsuccess. Therefore, a new approach to solve the problemon GRVs’ NUE is urgently needed. There should be somea principle that explores NUE within an overall plantbiology system and considers the co-regulation of plantgrowth, C and N metabolisms as a whole, rather thanfocusing specifically on N uptake and assimilation.

INTEGRATIVE NITRATE SIGNALING

Plant growth and development depend on N availability,and root N uptake is strongly influenced by changes ineither soil N availability or N demand of the whole plant.NO3

- is not only an essential nutrient for plants, butalso acts as signal molecule to regulate plant growth andN metabolism. Recently, understanding of the molecularmechanisms underlying plant adaptation to N deficiencyhas significantly progressed in the model plants: First,AtNRT1.1/AtNPF6.3 transporter and its functionalhomologs in other species fulfill a dual transport/sensing function, and function as transceptors (Ho et al.2009). Second, key components of N signaling pathwayshave been identified (Fig. 2) (Vidal et al. 2010; Gan et al.2012; Marchive et al. 2013; Liu et al. 2017; Jia andWiren 2020). Finally, plant growth-metabolism coordi-nation has been established, emerging connectionsbetween phytohormones (e.g., auxin, GA and ABA) andN signaling pathways (Vidal et al. 2013; Leran et al.2015; Li et al. 2018; Wu et al. 2020b).

Nitrate sensing

The Arabidopsis chl1-5 mutant caused by a null muta-tion of AtNRT1.1/AtNPF6.3, exhibits reduced NO3

-

uptake and NO3- responsive signaling, including short-

time (30 min) NO3--induced expressions of N-respon-

sive genes (e.g., AtNRT2.1), and lateral root development(Remans et al. 2006; Ho et al. 2009; Bouguyon et al.2015). However, the chl1-9 mutant is a weak mutantallele compared to the chl1-5 mutant, and exhibitsdefective NO3

- uptake, but no changes in primarynitrate responses (PNR) (Ho et al. 2009). In rice, theosnrt1.1b mutant also exhibits reduced expressionlevels of the NO3

--responsive genes, such as OsNIA1 andOsNIA2 (Hu et al. 2015). These results suggest thatNO3

- uptake and N responsiveness are separate pro-cesses, AtNRT1.1/AtNPF6.3 acts as a NO3

-sensor thattransduces external signal into the cells and triggersactivation of N signaling pathway (Ho et al. 2009;Bouguyon et al. 2015). Making AtNRT1.1/AtNPF6.3switch from low-affinity to high-affinity transporter isregulated by the phosphorylation status of the con-served amino acid residue Thr101 (Liu et al. 1999; Liuand Tsay 2003). Under low NO3

- conditions, the Thr101residue of AtNRT1.1/AtNPF6.3 is phosphorylated by theCBL9 (Calcineurin B-like protein 9)-CIPK23 (CBL-in-teracting protein kinase 23) protein complex, which inturn modulates AtNRT1.1/AtNPF6.3 activity, therebytriggering a low N stress response (Ho et al. 2009;Bouguyon et al. 2015). From the determination of thecrystal structure of AtNRT1.1/AtNPF6.3, the histidine

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residue 356 (His356) is important for NO3- binding,

and dephosphorylated AtNRT1.1/AtNPF6.3 tends toform a dimer that is required for the affinity switch fromhigh to low transport activity. Phosphorylation of theThr101 residue either influenced its functional dimer-ization or altered its structural flexibility, thus affectingboth NO3

- transport activity and signal transduction(Parker and Newstead 2014; Sun et al. 2014b). It hasnot been conclusive about the NO3

- sensor in rice now.A recent study shows that an AtNRT1.1 homologue

OsNRT1.1B can recruit an E3 ligase NBIP1 to promotedegradation of SPX4 via 26S proteasome pathway whenplants are treated with 10 mM NO3

-, thus releasingOsNLP3 protein, an ortholog of Arabidopsis AtNLP7, intothe nucleus to activate downstream responses (Hu et al.2019) (Fig. 2).

The abi2-2 mutant exhibits the reduction of NO3--

induced expression of AtNRT2.1 and lateral root growth,suggesting that ABI2, a key regulator of ABA signalling,is required for NO3

- sensing and signaling (Leran et al.

Fig. 1 Illustration of some reported N metabolism related genes in several crop species. OsNPF2.4, OsNPF6.1, OsNPF7.2, OsNPF7.4,OsNPF6.5/NRT1.1B, OsNPF6.3/NRT1.1A, OsNRT2.1, and OsNRT2.3a are involved in nitrate uptake. OsAMT1;.1 and OsNPF8.20/PTR9NPF2.2are involved in ammonium uptake. OsNPF2.2, OsNPF2.4, OsNPF6.5/NRT1.1B, OsNPF6.3/NRT1.1A, OsNPF7.1 and OsNRT2.3b function inroot-to-shoot nitrate translocation. OsNPF6.1, OsNPF7.1, OsNPF7.3 and OsNPF8.20/PTR9 participate in nitrate allocation during leafsenescence. The rice OsNR2 and OsGS1;1, OsGS2 take parts in nitrate reduction and ammonium assimilation processes, respectively. All thegenes demonstrated to improve plant NUE and grain yield have been marked by blue. The wheat GS2 homologue TaGS2-2, the maize GS1homologues ZmGS1-3/ZmGln1-3 and ZmGS1-4/ZmGln1-4 are shown to increase N uptake and assimilation, and consequently enhancegrain yield. HATS, high-affinity transport system; LATS, low-affinity transport system

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2015). It is found that ABI2 can interact with anddephosphorylate both CBL1 and CIPK23, which in turninhibits activity of the CBL1–CIPK23 complexes, therebyregulating AtNRT1.1/AtNPF6.3-dependnent NO3

- sens-ing and signaling (Fig. 2). In addition, the effects of NO3

–

on lateral root development are reduced but not com-pletely abolished in the ABA-deficient and ABA-insen-sitive mutants (e.g., abi4-1, abi4-2 and abi5-1) (Signoraet al. 2001). It is known that ABI2, can also be regulatedby various biotic and abiotic stresses (Rodriguez et al.1998), suggesting that the crosstalk between ABA andNO3

– signaling provides a new mechanistic insight into

the N metabolism in response to various environmentalstresses.

In Arabidopsis, CIPK8 is a sequence homolog ofCIPK23, and the cipk8-1 mutant is defective in low-affinity phase of PNR (Hu et al. 2009). The expression ofCIPK8 is rapidly induced by NO3

- supply, and thatpositively regulates expressions of the NO3

- responsivegenes. However, there is no evidence that AtNRT1.1/AtNPF6.3 can be phosphorylated by the CBLs–CIPK8protein complex. In addition, CIPK23 interacts directlywith both AtAMT1;1 and AtAMT1;2, and CBL1–CIPK23-mediated phosphorylation of AtAMT1;2 causes inacti-vation of NH4

? transport (Straub et al. 2017). Taken

Fig. 2 Nitrate signaling in the integrative regulation of N metabolism and plant growth. In response to low N concentrations, NPF6.3 canswitch to high-affinity status after the phosphorylation by CBL1/9–CIPK23 complex, which is inhibited by phosphatases ABI2. Inresponse to high external N level, the dephosphorylated NPF6.3 tend to form a dimer with low-affinity activity. The dual-affinity nitratetransporter NPF6.3 also act as a nitrate sensor to trigger calcium signals. The calcium-dependent protein kinase CPK10/30/32 canphosphorylate NLP7 and cause its nuclear retention, then consequently regulates N-responsive N assimilation and plant growth. In rice,the NPF6.3 ortholog OsNRT1.1B could recruit NBIP1 (an E3 ligase) and degrade SPX4 via 26S proteasome, thereby releasing the riceortholog OsNLP3 protein into the nucleus. TCP20 is a key component in regulating root foraging progress. The interaction between TCP20and NLP6/7 plays key roles in the expression regulations of target genes. In addition, G protein is also required for nitrate sensing andresponses. Some a receptor like kinase (RLK) interact with Ga subunit and then release the freely Gbc dimer, which can modulate thedownstream effectors (i.e., MADS family transcription factor). miR393-AFB regulatory module integrate auxin signaling pathway intonitrate responsive plant growth. Besides that, NLP7 transcription factor can directly induce the expressions of auxin biosynthetic geneTAR2 and auxin efflux gene PIN7, consequently leading to changes of cellular auxin levels. In the absent of nitrate, NPF6.3 facilitates auxinaway from the lateral root primordium tip and inhibit the lateral root outgrowth

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together, these results suggest that the CBLs–CIPK23protein complexes appear to occupy a prime position inregulating N homeostasis in plants.

In addition to transporters and channels, severalreceptor-like kinases (e.g., CLV1, GHR1 and HPCA1)have been shown to function as receptors or sensors totransduce the extracellular signals into multiple cellularresponses in Arabidopsis (Trotochaud et al. 2000; Sierlaet al. 2018; Wu et al. 2020a). The maize FEA2 (FAS-CIATED EAR2), an ortholog of receptor-like kinase CLV2,is shown to interact directly with the maize G-protein asubunit CT2 (COMPACT PLANT2), which in turn trans-mit external signals into the cells, thereby controllingproliferation of the stem cells in shoot apical meristem(Bommert et al. 2013). This result suggests that recep-tor-like kinases play an important role in signal per-ception and transduction via integrating with theheterotrimeric G proteins (Xu et al. 2016b). Further-more, a grain number quantitative trait locus DENSEAND ERECT PANICLE1 (DEP1), a gene encoding the riceG-protein c subunit (Gc), has been shown to be involvedin N-mediated growth responses (Huang et al. 2009;Sun et al. 2014a). Loss-of-function dep1-32 mutantexhibits N-insensitive responses, with respect to plantheight, tiller numbers and grain numbers, suggestingthat DEP1 is a positive regulator of N-promoting plantgrowth. Interestingly, the rice plant carrying the gain-of-function dep1-1 allele exhibits N-insensitive growthresponses coupled with increased N assimilation.Resultant semi-dwarf plants resist lodging and improvegrain yield with reduced N fertilizer inputs (Sun et al.2014a). DEP1 can interact physically with both Ga andGb subunits, either reduced activity of Ga or increasedactivity of Gb causes N-insensitive vegetative growth.These results suggest that G proteins are required for Nsensing and signaling pathway (Sun et al. 2014a; Xuet al. 2016b). A new study has demonstrated that DEP1protein acts as a co-factor of MADS-domain transcrip-tion factor OsMADS1 through the protein interaction,and co-regulates their common downstream genes toimprove grain yield in rice (Liu et al. 2018). Interest-ingly, the Arabidopsis MADS-domain transcription factorANR1 is firstly demonstrated to regulate lateral rootbranching and growth in response to NO3

- (Zhang andForde 1998). These results suggest that Gc-MADS reg-ulatory module may be an unique signaling pathwaywith key roles in N sensing and signal transduction(Fig. 2).

Local nitrate signaling

N availability often limits plant growth and crop pro-duction because of spatial and temporal fluctuations of

its concentration in the soil. Plants have developed amyriad of adaptive mechanisms allowing them to senseboth external N availability and their own nutritionalstatus, and quickly respond to the fluctuations of itsavailability. Typical responses to low N stresses includeincreased activity and affinity of N transporters,enhanced lateral root growth, thus promoting soilresource acquisition by plant roots. The NO3

- fluctua-tions has a great effect on both short-term and long-term gene expressions at the whole-plant level, includ-ing those related to NO3

- uptake (e.g., AtNRT1.1,AtNRT2.1 and AtNRT2.2), N assimilation (e.g., AtNIA1,AtNIA2 and AtNiR) and N-responsive transcription fac-tors (Wang et al. 2004, 2007; Scheible et al. 2004; Krouket al. 2010b; Alvarez et al. 2012).

Significant advances have been made during therecent period concerning the molecular mechanismsabout N-associated regulations of chromatin modulationand gene expression (Gaudinier et al. 2018; Alvarezet al. 2019; Wu et al. 2020b). A series of NO3

-

responsive transcription regulators and their interac-tions with the target PNR genes (e.g., AtNRT2.1 andAtNIA1) have been verified (Castaings et al. 2009;Konishi and Yanagisawa 2013; Marchive et al. 2013;Guan et al. 2017). The expressions of LBD37/LBD38/LBD39 genes are induced by NO3

- treatment, which inturn repress the expressions of a subset of NO3

-

assimilation genes (Rubin et al. 2009). The expressionlevels of NIGT1/HRS1 are positively regulated by NO3

-

supply in both Arabidopsis and rice. The ArabidopsisAtNIGT1 can repress an array of N starvation responsivegenes (Medici et al. 2015; Kiba et al. 2018), and riceOsNIGT1 protein can bind to its own promoter to fulfillthe auto-repression mechanism in response to N avail-ability (Sawaki et al. 2013). Using TARGET (transientassay reporting genome-wide effects of transcriptionfactors) and ChIP-seq (chromatin immunoprecipitationsequencing) analyses, the transcription factors bZIP1and SPL9 have been identified to play the key roles inregulating rapid NO3

--responsive genes (Krouk et al.2010b; Para et al. 2014; Vidal et al. 2015). Theexpressions of TGA1 (TGACG MOTIF-BINDING FACTOR 1)and TGA4 involved in regulating N metabolism arepositively correlated with N supply (Alvarez et al. 2014;O’Brien et al. 2016), and NO3

--induced expression ofTGA1 is found to be dependent upon a phospholipase C(PLC)-calcium signaling downstream of AtNRT1.1/AtNPF6.3 (Riveras et al. 2015). Strikingly, the TGA1–CIPK23 interaction may suggest that NO3

--mediatedactivation of TGA1 is in an AtNRT1.1/AtNPF6.3-depen-dent manner (Yazaki et al. 2016).

It has been shown that the NO3--unresponsive

transcription factors NLP6, NLP7 and TCP20 are key

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regulators of PNR, and regulate the expressions of somedownstream NO3

--responsive transcription factors(Fig. 2). Under low N conditions, the TCP20-NLP6/7heterodimers accumulate in the nucleus, bind to thepromoter regions of some N assimilation genes (e.g.,AtNRT2.1 and AtNIA1), thus promoting their expression.In addition, the TCP20-NLP6/7 heterodimers are alsorequired for down-regulation of the cell-cycle progres-sion gene CYCB1;1 and inhibition of root growth underlow N conditions (Guan et al. 2017), suggesting thatTCP20–NLP6/7 interactions coordinate the balancebetween N acquisition and root growth in response toNO3

- availability. Recent studies have shown that NLP6and NLP7 could be retained within the nucleus, and thisretention in response to NO3

- supply occurs withinminutes (Marchive et al. 2013; Guan et al. 2017).Moreover, the nuclear retention of NLP7 protein isshown to correlate with the CPK10/CPK30/CPK32-de-pendent phosphorylation. All these results give rise to akey concept of NO3

- CPKs–NLP7 signaling in centralnutrient-growth network (Castaings et al. 2009; Marc-hive et al. 2013; Liu et al. 2017) (Fig. 2). Besides that,another NO3

--unresponsive transcription factorNITRATE REGULATORY GENE2 (NRG2) can bind to thePNR genes’ promoters and regulate their expressionlevels (Xu et al. 2016a). Although NRG2 is demonstratedto directly interact with NLP7, the detailed significanceof this interaction in response to N availability stillremains unclear.

The recent discoveries linking NO3- and hormone

signaling have advanced our understandings about themolecular mechanism underlying the plant develop-mental plasticity in response to NO3

- availability(Fig. 2). Auxin-mediated root system patterning play animportant role in NO3

--responsive root growth. Usingenhanced yeast one-hybrid assays, a N-responsivetranscriptional regulatory network has been established(Gaudinier et al. 2018), including previously unidenti-fied transcription factors, for example, ARF9 regulatesthe expressions of PNR genes (e.g., GLN2 and ASN20),and ARF18 mediates the expressions of some new-identified target genes (e.g., NRT2.4 and AMT1;2).Indeed, AtNRT1.1/AtNPF6.3 can function as an auxininflux facilitator (Krouk et al. 2010a). The phospho-mimetic AtNRT1.1/AtNPF6.3T101D is shown to facilitateauxin flux and fast lateral mobility, whereas nonphos-phorylated AtNRT1.1/AtNPF6.3T101A exhibits low auxintransport capacity and causes the accumulation of auxinin the tips of lateral roots (Zhang et al. 2019), suggestingthat NO3

- starvation-induced phosphorylation of T101promotes AtNRT1.1/AtNPF6.3 vesicle cycling and facil-itates auxin flux, thus regulating auxin-mediated lateralroot growth. Genome-wide transcriptional analysis

show that the expression levels of auxin biosyntheticgene TAR2 and auxin efflux gene PIN7 are induced byNLP7 (Liu et al. 2017). The tar2 mutant exhibitsreduced auxin accumulation in lateral root primordiaand inhibition of lateral root formation under low Nconditions (Ma et al. 2014). In addition, overexpressionof CmANR1, a chrysanthemum ANR1 homologous,influences lateral root development by promoting auxinbiosynthesis and transport (Sun et al. 2018). Interest-ingly, NO3

--induced expression of auxin receptor geneAFB3 is observed in root tips, which can be subse-quently post-transcriptionally repressed by miR393, amicroRNA that is induced by NO3

- and repressed by Nmetabolites generated by NO3

- reduction and assimi-lation (Vidal et al. 2010). The NO3

--responsive miR393-AFB3 module and its downstream transcription factorNAC4 are capable to regulate lateral root growth inresponse to NO3

- supply (Vidal et al. 2013), indicatingthat miR393-AFB3-NAC4 is a unique N-responsivemodule that controls plant architecture in response tochanges in N availability.

There are also many crosstalk between N and ABA.AtNRT1.1/AtNPF6.3 transport activity was shown to becontrolled by CBL1/9–CIPK23 protein complex. ABI2can interact with and dephosphorylate both CBL1 andCIPK23, which in turn inhibits CBL1–CIPK23-mediatedsignaling, thereby regulating AtNRT1.1/AtNPF6.3-de-pendnent NO3

- sensing and signaling (Fig. 2). ABI2 is akey regulator of ABA signaling, and it is activated byvarious biotic and abiotic stresses (Rodriguez et al.1998). The abi2-2 mutant exhibited the reduction ofNO3

--induced expression of AtNRT2.1 and lateral rootgrowth, suggesting that ABI2 is required for NO3

-

sensing and signaling (Leran et al. 2015). Beside that,ABA also plays a crucial role in the inhibitory effect ofhigh NO3

- provision (C 10 mM) on lateral root (LR)development. Three ABA-insensitive mutants, abi4-1,abi4-2 and abi5-1, and all the ABA synthesis mutantsshowed significantly reduced but not completely abol-ished inhibition by NO3

-, implying that there are ABA-dependent and ABA-independent pathways to mediatethe NO3

- effects on LR elongation (Signora et al. 2001).All these crosstalk between NO3

- uptake and ABA sig-naling provides a new mechanistic insight into the ionicbalance between NO3

- and other nutrients in responseto various environmental stresses.

Using mRNA sequencing, genome-wide RNA poly-merase II (RNPII), ChIP and DNase sequencing data sets,the relationships between RNPII occupancy and chro-matin accessibility in response to NO3

- have beenestablished in Arabidopsis roots (Alvarez et al. 2019).The chromatin factor HNI9 (high nitrogen-insensitive9), a conserved component of RNA polymerase II

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complex, is shown to suppress the AtNRT2.1 expressionvia histone H3 lysine 27 tri-methylation (H3K27me3)modifications under high N conditions (Widiez et al.2011). The sdg8-5 mutant that lacks histone methyl-transferase SDG8 exhibits not only impaired regulationof ASN1 promoter (Thum et al. 2008), but also impairedH3K36me3 modifications at genomic loci functionallyrelevant to NO3

- uptake, resulting in reduced metabolicand developmental responses to N treatments (Li et al.2020). A new study shows that the rice AP2 transcrip-tion factor NITROGEN-MEDIATED TILLER GROWTHRESPONSE 5 (NGR5) is positively regulated by N supply,and it interacts directly with PRC2 complex and conse-quently alters the genome-wide H3K27me3 pattern,thus regulating the expressions of N-responsive genes inresponse to N availability (Wu et al. 2020b).

Systemic nitrate signaling

Plants modulate the efficiency of root N acquisitioncoupled to the shoot N demand. Indeed, NO3

- functionsas not only a local signal molecule, but also a long-dis-tance systemic signal (Wang et al. 2004; Ruffel et al.2011). Under low N conditions, the Arabidopsis CLEgenes that encode CLAVATA3/ENDOSPERM SUR-ROUNDING REGION-related peptides are induced inroot pericycle cells, the products of which diffuse intophloem companion cells, then bind to and activate leu-cine-rich repeat receptor protein kinase CLAVATA1(CLV1). Cell-to-cell communication medicated by CLE-CLV1 signaling plays an important role in inhibiting theoutgrowth of lateral root primordia in response to NO3

-

(Araya et al. 2014a, b). Previous studies have shownthat N starvation-induced CEP (C-terminally encodedpeptide) in the roots acts as a long-distance systemicsignal. The root-to-shoot translocated CEP is recognizedby the repeat receptor kinases CEPR1 (CEP receptor 1)and CEPR2 (Tabata et al. 2014), consequently resultingin production of the non-secreted polypeptides CEPD1(CEP downstream 1) and CEPD2 in the shoots. Resultantpeptides act as second mobile signals, which are furthertranslocated to the roots via the phloem and induceAtNRT2.1 expression in both deprived and non-deprivedroots (Ohkubo et al. 2017). In addition, the peptidesCEPD-like 1 (CEPDL1) and CEPDL2 share high sequencesimilarity with CEPD1. The expression of CEPDL2 isreversibly down-regulated by the shoot N status, andshoot-to-root translocated CEPDL2 cooperates withCEPD1 and CEPD2 to systemically regulate root NO3

-

acquisition (Ota et al. 2020). These findings reveal thatlong-distance peptide signaling pathways play key rolesin the coordination between N acquisition and thewhole plant demand (Fig. 3).

TCP20 interacts directly with NLP6/NLP7, and pro-motes the expressions of AtNRT2.1 and other NO3

-

metabolism genes (Guan et al. 2017). Unlike the wild-type plant and nlp7 mutant, the tcp20 mutant exhibitsimpaired root foraging on heterogeneous NO3

- media insplit-root plates (Guan et al. 2014), suggesting thatthere is a TCP20-mediated unknown systemic signalrequired for root nutrient foraging response (Fig. 3). Inaddition, root-derived cytokinin (CK) is found to adjustshoot meristem homeostasis and plant architecture inresponse to changes in N availability (Landrein et al.2018). The expression of IPT3, a gene encoding the keyenzyme of CK biosynthesis, is up-regulated in responseto increasing N supply, thus causing the rapid increaseof CK levels in the roots. Root-synthesized CK istranslocated to the shoots, and promotes N assimilationand shoot growth (Ruffel et al. 2011). AtABCG14 acts asa transporter that controls root-to-shoot translocationof trans-zeatin (tZ)-type CK in Arabidopsis (Zhang et al.2014), the atabcg14 mutant not only exhibits theremarked reduction of CK levels in the xylem andretardation of shoot growth, but also represses systemicN-demand signaling (Poitout et al. 2018), suggestingthat mobile CK co-regulates root N acquisition and shootN demand in response to different N conditions. Takentogether, engineering or breeding for modulating activ-ity of the mobile signals provides a new strategy toimprove NUE in crops.

It has long been known that N acquisition is tightlycoupled with photosynthetic C fixation. Light promotesroot growth and NO3

- uptake, whereas the hy5-526mutant that lacks a bZIP transcription factor ELON-GATED HYPOCOTYL5 (HY5) abolishes light-dependentroot growth and NO3

- uptake in Arabidopsis. Furthergraft experiments demonstrated that HY5 is a mobilesignal that mediates coupling of C fixation and NO3

-

uptake (Chen et al. 2016) (Fig. 3). This study impliesthat the systemic signaling should not focus on N sig-naling specifically, but also take C fixation and plantgrowth into account because of their indivisibilitywithin an overall plant system.

PLANT GROWTH-METABOLISM COORDINATION

Plants use sunlight energy to convert carbon dioxide(CO2) into photoassimilate (e.g., starch and sugars) inthe shoots, and take up N by the roots. C fixation pro-vides the C skeletons needed for NO3

- assimilation(Boussadia et al. 2010). Interaction between C and Nmetabolisms in plants is important for efficient assimi-lation of these two major nutrients and thus to maxi-mize plant growth and grain yield (Zheng 2009).

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C/N metabolism and allocation coordination

The coordination of C and N metabolites (also refer to asC/N ratio or C/N balance) is of great importance foroptimal plant growth and crop productivity, except tothe C and N assimilation individually. Alterations inphotosynthesis and sugar metabolism greatly influenceN assimilation and plant growth. Elevated pCO2 cancause the increase in N uptake, reduction and allocation(Kruse et al. 2003). Overexpression of the Rubisco (rbcS)gene leads to an elevated N storage in rice leaves(Suzuki et al. 2007), whereas reduced Rubisco activityresults in an inhibition of N metabolism (Matt et al.2002). In addition, N metabolism defective plantsexhibit abnormal C/N balance, for example, the GS2-

cosuppressed plants have a higher concentration ofsoluble proteins and a lower concentration of carbohy-drates at the seedling stage, whereas the converse at thetillering stage (Bao et al. 2015b). When compared withthe wild-type plant, the nia1 nia2 mutant exhibits dif-ferent C and N status in different plant tissues, and itshows a lower level in leaves and an equal level in rootsor flower buds (Santos-Filho et al. 2014). All theseresults suggest that plants have developed a sophisti-cated system to spatiotemporally coordinate C and Nallocations.

In the photosynthetic leaves, C assimilation (e.g.,Calvin Cycle) produces triose phosphate that are usedfor the synthesis of either carbohydrates or ketoacids.2-OG produced by the tricarboxylic acid cycle (TCA

Fig. 3 A simplified model of systemic nitrate signaling in root foraging responses. Local N deprivation can trigger the production of CLEpeptides and CEP peptides. CLE peptides are recognized by the nearby CLV1 receptor resulting in restriction of lateral root growth.Meanwhile, CEP peptides are translocated from roots to shoots, and perceived by a kind of leucine-rich repeat receptor kinases (LRR-RKs)named as CEP receptors (CEPR), and then leading to generation of a secondary signal polypeptides named as CEPD (CEP downstream) inshoots, that further move back to the roots and promote nitrate uptake and root growth. As the homologue of CEPD, CEPDL (CEPD like)also can translocate from shoots to roots, contributing to regulate root nitrate acquisition cooperatively. In addition, HY5 is a lightstabilized transcription factor, which can induce the expression of TPS1 gene in shoots. As a systemic signal, shoot-to-root mobile HY5protein will induce the expression of NRT2.1 gene in roots to stimulate the ability of nitrate absorption. Although there is no clue aboutthe systemic signal, TCP20 has been demonstrated to play key roles in nitrate systemic signaling pathway by split-root assays

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cycle) is imported into chloroplasts, where it partici-pates in reception of NH4

? for the amino acid synthesis,and plays a key role in mirroring C/N balance (Huergoand Dixon 2015). In addition, trehalose-6-phosphate(T6P) not only regulates plant growth and development,but also act as a signal and a negative feedback regu-lator of cellular sucrose level (Wahl et al. 2013; Yadavet al. 2014). T6P can directly divert carbohydrate intothe C skeletons for amino acid synthesis, and accelerateN assimilation via post-translational activation of NRenzyme (Figueroa et al. 2016). To meet the demand ofboth carbohydrates and amino acids, C partitioning issubject to a sophisticated regulation, and T6P functionsindispensably in this coordination of C and N metabo-lism (Lawlor and Paul 2014).

C/N responses coordination

The coordinated regulation of C and N partitioningbetween sources (e.g., leaves) and sinks (e.g., roots andgrains) is important, because it enables plants to regu-late growth and metabolic responses under differentenvironmental conditions. A number of positive andnegative regulatory elements have been identified toregulate the C/N response in higher plants, includingGS2, NR, aquaporin and glutamate receptor GLR1.1(Kang and Turano 2003; Bao et al. 2015b; Gao et al.2018). In the presence of C, the putative glutamatereceptor AtGLR1.1 is negatively affected by sucrose,which causes decreased HXK1 transcript accumulationand elevated ABA levels, thus inhibiting the germinationof antiAtGLR1.1 seed. In contrast, AtGLR1.1 is positivelyaffected by N, and the germination can be restored uponco-incubation with NO3

- (Kang and Turano 2003),suggesting that AtGLR1.1 functions as a co-regulator ofplant growth, C and N metabolism by modulating ABAresponse. ABI1, encoding a type 2C protein phosphatasein ABA signal transduction pathway (Moes et al. 2008),is identified as the corresponding gene of the C/N-nu-trient response mutant cni2-D (Lu et al. 2015). Thetransgenic plants overexpressing ABI1 is found to beinsensitive to high C/low N stress, whereas the abi1-2mutant was hypersensitive, suggesting that ABI1 playsan essential role in the plant C/N response.

GS and NR are two important enzymes during nitrateassimilation, both of which are posttranslationallymodified by phosphorylations to influence their cat-alytic activities (Moorhead et al. 1996; Riedel et al.2001). NR is found to be the rate-limiting step for Nacquisition and C fixation. The expression levels of NRgenes and its corresponding enzyme activity are highlyregulated by multiple phytohormonal and environmen-tal factors, such as circadian rhythms, CO2, N supply and

so on (Ferrario-Mery et al. 1998; Yanagisawa 2014). NRcontains three catalytic domain, Mo-molybdopterin andinterface (Mo-MPT) domain, cytochrome b (cyt b)domain, and FAD and NADH domain, the hinge 1 andhinge 2 regions are localized between two adjacentdomains and joined them together (Campbell andKinghorn 1990). NR is phosphorylated by SnRK1 kinaseon the conserved serine residue (Ser534 in Arabidopsis)and subsequently bound by 14-3-3 protein to inhibit theNR activity (Su et al. 1996; Lambeck et al. 2012). Thephosphorylation of NR may be associated with optimalresponses to decrease in CO2 levels, increase in cytosolicpH, and various types of environmental stresses. Incontrast, another kind of kinase, mitogen-activatedprotein kinase 6 (MPK6) phosphorylates the hinge 2region of NR at serine residue Ser627, which causes anincrease of NR activity about 2.5-fold (Wang et al. 2010).The E3 ubiquitin ligases ATL31 and ATL6 have beenshown to regulate multiple responses to C/N nutrientavailability by triggering the degradation of 14–3-3protein (Sato et al. 2011; Yasuda et al. 2014; Xu et al.2019). In the ATL31-14-3-3 regulatory module, thephosphorylation of ATL31 is performed by three CBL-interacting protein kinases, CIPK7, CIPK12, and CIPK14,and these phosphorylations are important for 14-3-3binding ability, which in turn destructs 14-3-3 stabilityand then controls plant C/N-nutrient response (Yasudaet al. 2017). Furthermore, 14-3-3 proteomic analysishave shown that some key enzymes involved in Cmetabolism, such as sucrose synthase and invertase, arealso the targets for 14-3-3 protein (Alexander andMorris 2006; Gao et al. 2014). Thus, 14-3-3 proteinplays an important role in coupling of C and Nassimilation.

Growth-metabolism coordination signaling

Among all essential mineral elements, nitrogen (N) isrequired in the largest amount and thus is often a lim-iting factor, so the high-yielding crop production reliesheavily on N fertilization. For efficient N uptake fromsoil, plants have developed sophisticated and coordi-nated mechanisms to regulate plant growth, C fixationand N assimilation. The growth and development ofplant architecture are systematically regulated by Nstatus (Luo et al. 2020), and the N acquisition by roots isalso modulated by C status. For example, the expres-sions of NRTs genes (e.g., NPF6.3/NRT1.1 and NRT2.1)and their transporter activities are regulated by both Nand C status (Tsay et al. 1993; Lejay et al. 1999; Zhuoet al. 1999; Krouk et al. 2006). As mentioned above, amovable HY5 protein plays an important role in thecoordination of plant growth, C and N metabolism in a

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whole-organismal level (Chen et al. 2016). In the shoots,light-activated HY5 promotes the expression of thosegenes involved in photosynthetic C fixation (e.g., PSY andTPS1) and N assimilation (e.g., AtNIA1 and AtNIA2),consequently increasing light fluence-promoted CO2 andNO3

- assimilation. In the roots, shoot-derived sucrosepromotes uptake of NO3

-, but sucrose-mediated acti-vation of AtNRT2.1 expression and NO3

- uptake isdependent upon shoot-to-root HY5 function (Chen et al.2016). This coupling is achieved via HY5-regulation of Cfixation in the shoots, and via sucrose-enhanced pro-motion of HY5-dependent N-uptake in the roots. Thus, amobile HY5 is essential to allow plants to maintainhomeostatic balance of C and N metabolism in responseto ambient light conditions, and modulating its activityprovides a new strategy to improve crop NUE. In addi-tion, the plant-specific Dof1 family proteins have beenshown to modulate C skeleton production, overexpres-sion of either maize ZmDof1 or rice OsDof2 couldimprove NUE and grain yield under low N conditions(Yanagisawa et al. 2004; Kurai et al. 2011; Iwamoto andTagiri 2016), suggesting that the yield-increasing effectsof Dof1 protein should be attributed to the synergyimprovement of C/N metabolism.

In an effort to overcome the yield ceiling of rice GRVs,‘‘Super Rice’’ in China has been proposed to achievehigher yield potential in contemporary conventionalbreeding. Since the late 1980s, an ideal plant architec-ture characterized by dense and erect panicle, with highyield and high photosynthetic efficiency, has beenselected for japonica rice breeding in China. QTL anal-ysis and map-based cloning demonstrated that DEP1 isassociated with each trait of reduced plant height,increased grain numbers and improved grain yield(Huang et al. 2009). The mutant dep1-1 allele, encodinga truncation of the G protein c subunit, confers reducedexpression of OsCKX2 and enhanced biosynthesis of CK,thus causing increases in grain numbers and rice yield.More importantly, the rice plants carrying the dominantdep1-1 allele exhibited N-insensitive phenotype coupledwith increased N uptake and assimilation, resulting inincreased yield at moderate levels of N supply (Sun et al.2014a). The DEP1 protein interacts directly withOsMADS1, a homolog of a NO3

--inducible ArabidopsisANR1, suggesting that manipulation of dep1-OsMADS1regulatory module may enable a novel breeding strategyfor future sustainable agriculture.

The high-yielding GRVs exhibit reduced NUE (Li et al.2018), which is mainly due to the accumulations ofDELLA proteins (DELLAs). To overcome the negativeeffect of DELLAs on N uptake and assimilation, sd1-containing indica rice varieties with higher rates of Nuptake were selected for genetic analysis. An allelic

variation at the GRF4 locus, a gene encoding the riceGROWTH-REGULATING FACTOR4 (GRF4) transcriptionfactor, is shown to enhance C and N assimilation, andcounteract the inhibitory effects of rice DELLA proteinSLR1 (Li et al. 2018). GRF4 is a positive regulator ofboth N-responsive plant growth and N assimilation, butits abundance decreases with increasing N. RNA-seq andChIP-seq analyses reveal that GRF4 binds to and acti-vates the expression of those genes involved in Nmetabolism (e.g., OsAMT1;1, OsGS1.2 and OsNR2), pho-tosynthetic C fixation (e.g., OsLhca1, OsTPS1 and OsS-WEET11), and cell proliferation (e.g., OscycA1;1 andOscdc20s-3), thus coordinating plant growth, C and Nassimilation. In addition, GRF4 interacts with SLR1 andGIF1 (growth regulating factor-interacting factor1), andthe interactions inhibits GRF4–GIF1 complex-mediatedgene activation. Therefore, the GRF4-DELLA balanceprovides molecular mechanisms for the previouslyunexplained and hugely important co-regulation of thebalance between N and C metabolism. More importantly,tipping the GRF4-DELLA balance towards increasedGRF4 abundance substantially increases NUE and grainyield of elite rice and wheat GRVs, without loss of yield-enhancing DELLA-conferred semi-dwarfism (Li et al.2018). The modulation of GRF4-DELLA regulatorymodule provides a relatively simple route for improve-ment of cereal crop NUE, a multigene trait whosegenetic complexity have previously made substantialimprovements difficult to achieve.

PROSPECTS

Crop production is heavily dependent on N fertilizersupply. To obtain high yields, N fertilizers have beenoverused, future agricultural sustainability demandsnew breeding strategies to cut fertilizer use in the high-yielding crop varieties. NUE can be divided into Nuptake efficiency, N assimilation efficiency and Nremobilization efficiency, which involve several biologi-cal processes and metabolic pathways of N uptake,assimilation, translocation, and remobilization (Mas-claux-Daubresse et al. 2010; Han et al. 2015). Strategiestargeting genes directly involved in N transport andmetabolism have been used to improve crop NUE andgrain yield. For example, introduction of the elite allelesof OsNRT1.1B and OsNR2 into the japonica rice varietiesenhance grain yield (Hu et al. 2015; Gao et al. 2019b).However, NUE is a complex trait, which is controlled bydifferent metabolic, developmental and environmentalresponse network interactions integrated over theentire life cycle of the plants. The principles of newapproaches should explore NUE within an overall plant

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systems biology context that considers the coordinationof plant growth, C and N metabolism as a wholeorganism responses to N availability, rather thanfocusing specifically on N assimilation alone. Recentstudies have shown that disconnecting plant growth(e.g., plant height and tillering) from N regulation byaltering either GRF4-DELLA, dep1-OsMADS1, or NGR5-DELLA module enables increased grain yield at low Nfertilizer inputs, without loss of yield-enhancing semi-dwarfism (Li et al. 2018; Liu et al. 2018; Wu et al.2020b) (Fig. 4). Therefore, modulating plant growth-metabolism coordination provides a novel breedingstrategy to cut fertilizer use in high-yielding GRVs.

In addition to inorganic NH4? and NO3

-, urea hasbecome the N fertilizer most widely used in agriculture,amounting to about half of total fertilizer consumption.Although high-affinity urea transporter DRU3 in

Arabidopsis and its orthologues in crops (e.g., ZmDUR3)have been demonstrated to mediate urea uptake intoroot cells (Zanin et al. 2014), it is believed that cropplants acquire most urea-N in the form of NH4

? throughmicrobial hydrolysis of urea in the soil (Nacry et al.2013). A new study showed that indica rice-enrichedroot microbiota were found to be more diverse, andcontain more genera with N metabolism functions, thanjaponica rice-enriched taxa (Zhang et al. 2019). Thus,manipulation of the coupling between plant genotypeand root microbiota community structure provides anew method to improve NUE in future.

Current advances in the understanding of themolecular mechanisms underlying N transport, sensingand signaling in model plants have been mostly per-formed under controlled laboratory conditions. In thefuture, it will, therefore, be key to stimulate the

Fig. 4 A sketch map of some functional genes in modulating plant growth and C–N metabolism coordination in rice. Rice GRVs (withmutant sd1 allele) exhibit decreases in plant height and crop NUE, which is conferred by the accumulation of DELLA protein. DELLA is animportant repressive protein in GA signaling, and it can be degraded after interacting with the GA receptor GID1. NGR5 acts as anotherGID1-interacting protein, consequently elevating its expression levels could partially inhibit the DELLA effects and further increase grainyield. Besides that, NGR5 is shown to up-regulate rice tiller outgrowth. GRF4 is a positive regulator of N-responsive plant growth, Cfixation and N assimilation. As a co-activator, GIF1 interacts with GRF4, and the interaction can be inhibited by rice DELLA protein SLR1.Therefore, tipping the GRF4-DELLA balance towards increased GRF4 abundance has been demonstrated to improve both plant NUE andgrain yield. DEP1 is firstly known as a high-yielding gene associated with notably increased grain number. As the gain-of function mutantallele, dep1 is also a positive regulator of crop NUE

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discovery of new regulatory networks in cereal GRVsgrown under field conditions. The identification of newgenes that coordinate plant growth and nutrientsmetabolism, as well as the development of new breed-ing strategies combined with field management prac-tices, could usher in a new green revolution.

Acknowledgements Due to limited space we apologize to par-ticipants whose excellent work not mentioned here. This workwas supported by grants from the National Key Research andDevelopment Program of China (2016YFD0100901), NationalNatural Science Foundation of China (31971916), and the YouthInnovation Promotion Association, Chinese Academy of Sciences(2019-100).

Compliance with ethical standards

Conflict of interest All the authors have no conflict of intereststatement.

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