The Arabidopsis CPSF30‐L gene plays an essential role in ......The Arabidopsis CPSF30-L gene plays an essential role in nitrate signaling and regulates the nitrate transceptor gene

The Arabidopsis CPSF30-L gene plays an essential role in nitratesignaling and regulates the nitrate transceptor gene NRT1.1

Zehui Li1*, Rongchen Wang2*, Yangyang Gao1*, Chao Wang1, Lufei Zhao1, Na Xu3, Kuo-En Chen4,

Shengdong Qi1, Min Zhang5, Yi-Fang Tsay4, Nigel M. Crawford6 and Yong Wang1

1State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong 271018, China; 2National Key Laboratory of Crop Genetic Improvement,

College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China; 3School of Biological Science, Jining Medical University, Rizhao, Shandong 276826,

China; 4Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan; 5College of Resources and Environment, Shandong Agricultural University, Tai’an, Shandong 271018, China;

6Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA

Author for correspondence:Yong Wang

Tel: +86 538 8243957Email: [email protected]

Received: 5 June 2017

Accepted: 4 July 2017

New Phytologist (2017)doi: 10.1111/nph.14743

Key words: Arabidopsis, CPSF30-L, nitrateassimilation, nitrate signaling, nitrate uptake,NRT1.1.

Summary

� Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitro-

gen availability. However, more underlying genes regulating the response to nitrate have yet

to be characterized.� We report here the identification of a nitrate regulatory mutant whose mutation mapped to

the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant,

induction of nitrate-responsive genes was inhibited independent of the ammonium conditions

and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L.� Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and

independently of NLP7 in response to nitrate. Analysis of the 30-UTR of NRT1.1 showed that

the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis

revealed that four nitrogen-related clusters were enriched in the differentially expressed genes

of the cpsf30mutant. Nitrate uptake was decreased in the mutant along with reduced expres-

sion of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid

content were enhanced in roots along with increased expression of several nitrate assimilatory

genes.� These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate sig-

naling in part by regulating NRT1.1 expression, thus adding an important component to the

nitrate signaling network.

Introduction

Nitrogen is an essential macronutrient for plant growth anddevelopment. Improving nitrogen-use efficiency (NUE) toreduce the loss of nitrogen into the environment is a worldwideproject for decreasing the use of nitrogen fertilizer and loweringits pollution of the environment. Most aerobic plants, includingmajor crops, absorb nitrate as the main nitrogen source (Craw-ford & Glass, 1998). Thus, nitrate is of great importance for agri-culture production. Nitrate is absorbed into roots through thenitrate transporters of the NRT1 and NRT2 families (Forde,2000; Li et al., 2007; Tsay et al., 2007). In the cell, nitrate isreduced to ammonium by nitrate reductase (NR) and nitritereductase (NiR) and then assimilated into amino acids throughenzymes such as glutamate synthase (GOGAT) and glutaminesynthase (GS) (Stitt et al., 2002; Vidal & Guti�errez, 2008).

In addition to serving as a nutrient, nitrate also serves as a sig-nal molecule which acts in both short- and long-term processes.The short-term response is referred to as the primary nitrateresponse, in which many genes are rapidly regulated after nitratesupply; for instance, some NRTs, NIAs and NiR are inducedwithin minutes of nitrate treatment (Wang et al., 2000, 2003,2004; Scheible et al., 2004; Guti�errez et al., 2007; Krouk et al.,2010c). The long-term action of nitrate is evident by its effectson plant growth and development, including effects on rootarchitecture, seed dormancy, flowering, circadian rhythms, andstomatal closure independent of ABA and auxin transport(Roenneberg & Rehman, 1996; Stitt, 1999; Alboresi et al., 2005;Walch-Liu et al., 2006; Wilkinson et al., 2007; Krouk et al.,2010b).

Previous reports on the nitrate signal functions have mainlyfocused on the primary nitrate response and root architecture(Alvarez et al., 2012; Forde, 2014; Medici & Krouk, 2014). Thefirst gene identified as playing an important role in nitrate signal-ing was a MADS box transcription factor ANR1, which regulates*These authors contributed equally to this work.

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lateral root branching in response to nitrate (Zhang & Forde,1998; Gan et al., 2012). NTR1.1 (CHL1), a dual-affinity nitratetransport gene, was subsequently found to act upstream of ANR1in the localized stimulatory response to nitrate (Remans et al.,2006; Gan et al., 2012). Furthermore, TCP20 works upstream ofNRT1.1 in the process of nitrate foraging (Guan et al., 2014). Asystems approach revealed that the nitrate-responsive AFB3-miR393 module and its downstream target NAC4 regulate rootsystem architecture in the presence of nitrate (Vidal et al., 2010,2013). NLP7 functions negatively in root growth under repletenitrate supply (Castaings et al., 2009). In addition, several geneshave been demonstrated to control root architecture in responseto other nitrogen forms or conditions, including NRT2.1, underthe condition of high nitrogen/carbon ratios (Little et al., 2005;Remans et al., 2006), HNI9 (IWS1), repressing the expression ofNRT2.1 at high nitrogen supply (Widiez et al., 2011), ARF8 inresponse to organic nitrogen (Gifford et al., 2008), and the CLE-CLAVATA1 peptide-receptor module under nitrogen-deficientconditions (Araya et al., 2014). Recently, HRS1 and HHO1, twoearly nitrate-regulated transcription factors, were identified tosuppress the primary root growth specifically in response tonitrate when phosphate is absent (Medici et al., 2015).

In recent years, a few important nitrate regulatory genes work-ing in primary nitrate response have been characterized. Thedual-affinity transporter NRT1.1 was reported to be a nitratesensor (Tsay et al., 1993; Liu & Tsay, 2003; Walch-Liu & Forde,2008; Ho et al., 2009; Wang et al., 2009). Its nitrate transportmechanisms have been revealed by crystal structure analyses(Parker & Newstead, 2014; Sun et al., 2014). CIPK8 andCIPK23, which are modulated by NRT1.1, function in primarynitrate response as a positive player for CIPK8 and a negativeplayer for CIPK23 (Ho et al., 2009; Hu et al., 2009). Moreover,CIPK23 phosphorylates NRT1.1 under low nitrate conditions(Ho et al., 2009). NRG2 was recently discovered to function as akey factor in nitrate regulation and to modulate the expression ofNRT1.1 (Xu et al., 2016). In addition, a few transcription factorshave been identified as being involved in the nitrate signalingpathway. NLP7 (NIN-like protein 7), which belongs to the RWP-RK transcription factor family, has been shown to be an essentialelement of the nitrate signaling (Castaings et al., 2009). Nitratetreatment can trigger nuclear retention of the NLP7 protein(Marchive et al., 2013). Furthermore, NLP6 and other NLP fam-ily proteins have been found to bind the nitrate-responsive cis-element of some nitrate-induced genes and to activate theirexpression (Konishi & Yanagisawa, 2013). The nitrate-regulatedtranscription factors LBD37/38/39, working as negative regula-tors, could repress many genes involved in NO3

� uptake andassimilation (Rubin et al., 2009). Using systems approaches,SPL9, TGA1, and TGA4 have been revealed as important regula-tors involved in primary nitrate response (Krouk et al., 2010c;Alvarez et al., 2014). The transcription factor bZIP1 was recentlycharacterized by TARGET and chromatin immunoprecipitation-seq (ChIP-seq) analyses as a key factor regulating rapidnitrate-responsive genes (Para et al., 2014; Vidal et al., 2015). Inthe process of evolution, plants have evolved complicated andsophisticated mechanisms to adapt to the changing nitrate

availability in the environment. The identification of thedescribed nitrate regulatory genes is just the start of unravelingthe nitrate signaling network.

In this paper, we isolated an Arabidopsis mutant Mut65 usinga forward genetic screen. The mutation was mapped to the geneCPSF30-L, which encodes a polyadenylation factor subunit. Fur-ther investigation showed that it is required for nitrate-dependentgene expression, and modulates nitrate content by regulatingnitrate transport and assimilation in plants. Molecular andgenetic assays revealed that CPSF30-L works upstream and regu-lates the expression of NRT1.1, while functioning independentlyof NLP7 in nitrate signaling.

Materials and Methods

Plant materials and mutant screen

Arabidopsis thaliana Columbia-0 ecotype was used as the wild-type (WT). The mutant lines chl1-13 (containing the NRP-YFPconstruct; original name Mut21) (Wang et al., 2009), chl1-5 (Hoet al., 2009), cipk8-1 (SALK_139697) (Hu et al., 2009), cipk23-3(SALK_036154) (Ho et al., 2009), and nlp7-4 (containing theNRP-YFP construct; original name Mut216) (Xu et al., 2016)were described previously. We used homozygous transgenic seedscontaining the NRP-YFP construct as the WT for observation offluorescent signals. cpsf30-2 with low fluorescence on the nitratemedium (10 mM KNO3) was screened by the assay that homozy-gous transgenic seeds treated with ethyl methanesulfonate (EMS)(Wang et al., 2009). Putative mutants were selfed and retested.Confirmed mutants were backcrossed to the transgenic WT twiceand homozygous lines were used for further analysis. The RNAivector was constructed with a 180 bp target sequence of CPSF30-L and transformed into the WT which contains the NRP-YFP(Supporting information Fig. S1). All the primers used are listedin Table S1(a).

Growth and treatment conditions

Seeds were germinated on a nylon mesh floating in liquid half-strength MS medium for 7 d and then the roots and shoots werecollected for nitrate accumulation, NR activity, and amino acidcontent assay, respectively. To test yellow fluorescent protein(YFP) fluorescence of transgenic plants in response to nitrate,plants were grown on a plate with 10 mM KNO3 or NH4NO3

(Xu et al., 2016) for 4 d followed by observation with the fluores-cence microscope (Nikon Eclipse Ti-S, Tokyo, Japan).

For gene expression tests using the quantitative PCR (qPCR)technique, seedlings were cultured hydroponically and treated aspreviously described (Xu et al., 2016). For nitrate treatment afternitrogen deprivation, plants were grown and treated as describedearlier except that they were transferred to nitrogen-free mediumfor 24 h on day 6. The roots and shoots were collected separatelyfor RNA extraction.

For the gene expression test under different nutrient starvationconditions, seedlings were grown on 10 mM NH4NO3 for 6 dfollowed by nutrient-deficient medium for 2, 3 and 4 d before

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collecting tissues for RNA extraction. For nitrogen starvation,NH4NO3 was removed from the medium. For the Pi-deficientmedium, 5 mM KCl was used in place of 5 mM KH2PO4 and1 mM MES was added into the medium to adjust the pH to 5.6(Wang et al., 2008).

qPCR analysis

Total RNA was isolated from Arabidopsis roots and shoots usinga total RNA miniprep kit (CWBIO, Beijing, China). cDNA syn-thesis was carried out using the RevertAid first-stand synthesissystem kit (Thermo Scientific, Waltham, MA, USA). TheUltraSYBR Green Mixture qPCR kit (CWBIO) was used in theqPCR reaction to determine the expression of relevant genesusing an ABI7500 Fast (Applied Biosystems, Waltham, MA,USA). TUB2 (At5g62690) was used as the internal referencegene. All the primers used are listed in Table S1(a).

Expression profile analysis by b-glucuronidase assay

A 2.9 kb genomic fragment immediately upstream of theCPSF30-L start codon was introduced into the vector pMDC162(Invitrogen). Transgenic plants carrying the ProCPSF30-L:: GUSconstruct were cultivated on half-strength MS medium for 7 d orin soil for 35 d and different organs were collected to detect theb-glucuronidase (GUS) activity, as described previously (Daiet al., 2014). The primers used for plasmid construction are listedin Table S1(a).

Subcellular localization

The full-length cDNA of CPSF30-L was introduced in framewith the GFP reporter gene at the C-terminal position (35S::CPSF30-L-GFP) in the binary vector pMDC83. The constructwas transformed into Arabidopsis (Col-0) plants and Nicotianabenthamiana, respectively, as described previously (Feng et al.,2008). The fluorescence was observed using a Leica TCS SP5IIconfocal microscope. The primers used for plasmid constructionare listed in Table S1(a).

Nitrate, amino acid content, and NR activity assay

Nitrate content was measured using a salicylic acid method asdescribed previously (Xu et al., 2016). Amino acid content wasmeasured as described by amino acid content kit (Solarbio,Beijing, China) and NR activity was detected by an NR activitykit (Solarbio). Nitrate uptake ability was measure using a 15N-labeled experimental method as described previously (Wang &Tsay, 2011).

Transcriptome analysis

The transcriptome analysis was performed using RNA sequenc-ing technology. Total RNA was isolated from the roots of 7-d-old seedlings grown on 2.5 mM ammonium succinate followedby 10 mM KNO3 or KCl treatment for 2 h. For each sample,

two biological replicates were used. The libraries were con-structed by NEBNext Ultra RNA Library Prep Kit for Illuminaand detected in an Agilent 2100 Bioanalyzer. HiSeq 2500 (Illu-mina, San Diego, CA, USA) technology was used for RNAsequencing. After filtering adapter, poly-N, and low-qualityreads, the effective data were mapped with the Arabidopsis (Ara-bidopsis thaliana) TAIR 10 reference genome TOPHAT2 (Lang-mead et al., 2009). Fragment per kb per million reads (FPKM)was used to estimate the abundance of the transcripts. The EDGERpackage (http://www.r-project.org/) was used to identify differen-tially expressed genes (DEGs) across treatments. We identifiedgenes with a fold change ≥ 2 and a false discovery rate(FDR) < 0.05 as significant DEGs which were then subjected toenrichment analysis of GO functions. GO functional annotationsof the data were determined using PANTHER (http://www.pantherdb.org/pathway/) (Mi et al., 2013). The RNA-seq data discussedin this article have been deposited in the National Center forBiotechnology Information database (www.ncbi.nlm.nih.gov/sra;accession number SRP111307).

Results

Isolation of the nitrate regulatory mutant Mut65

To explore new players involved in nitrate regulation inArabidopsis, we performed a screen for mutants defective innitrate signaling. The transgenic plants containing a nitrate-inducible promoter (NRP) fused to a YFP show much strongerfluorescence in the presence of nitrate than in the absence ofnitrate (Wang et al., 2009; Xu et al., 2016). The M2 populationfrom transgenic plant seeds mutagenized by ethyl methanesul-fonate were screened. A mutant named Mut65 displayed lowerfluorescence in roots than in the WT when grown on KNO3

medium (Fig. 1a). After twice backcrossing with WT, the nextgeneration consistently showed the same low fluorescence pheno-type, indicating that this mutant may harbor one or more mutag-enized genes involved in nitrate signaling. Based on this weakfluorescence phenotype, the mutation was mapped to At1g30460(CPSF30) with a G-to-A mutation in the first exon, whichresulted in a conversion of Gly to Arg at position 126 (Fig. 1b).

The product of the CPSF30 gene has been characterized as a28 kDa subunit of a cleavage and polyadenylation specificity fac-tor (Delaney et al., 2006; Addepalli & Hunt, 2007; Zhang et al.,2008). The CPSF30 gene also encodes a larger, 65 kDa protein.The 28 kDa protein (indicated as CPSF30-S in Fig. 1c–e) pos-sesses three characteristic CCCH zinc finger motifs and acts as anRNA-binding protein and an endonuclease (Addepalli & Hunt,2007). The 65 kDa protein (indicated as CPSF30-L in Fig. 1c–e)contains an additional YTH domain along with the three zincfinger motifs. Previously, an Arabidopsis cpsf30 mutant, oxt6, wasisolated and found to have enhanced oxidative stress tolerance,which could be reversed by expressing the 28 kDa protein alone(Zhang et al., 2008). To determine which of the two proteinsfunctions in nitrate signaling, the cDNAs encoding the smallerand larger forms of CPSF30 driven by the 35S promoter weretransformed into the Mut65 mutant. Three transgenic lines for


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each form (Fig. S2a,b) and 50 seedlings for each line were investi-gated for the fluorescence strength. The results showed thatCPSF30-L could rescue the weak fluorescence phenotype; whileCPSF30-S could not (Fig. 1d,e). We constructed an RNAi line ofCPSF30 (RNAi-L5-1) with 49% expression of CPSF30-L, whilethe expression of CPSF30-S was not significantly changed(Fig. S2c). The RNAi line displayed decreased YFP abundance,similar to that of Mut65 in the presence of nitrate (Fig. S2d,e).These results indicate that CPSF30-L, encoding the 65 kDa pro-tein, is responsible for the weak fluorescence phenotype and thusnitrate regulatory defect in cpsf30-2. Therefore, we renamedMut65 as cpsf30-2.

The cpsf30-2 is defective in the primary nitrate response

To determine if the 65 kDa protein encoded by CPSF30-L isinvolved in the primary nitrate response of endogenous genes,expression of three known nitrate-responsive genes, including

Nitrate Reductase 1 (NIA1), Nitrite Reductase (NiR), and high-affinity nitrate transporter gene NRT2.1, was examined. Asshown in Fig. 2(a), the relative expression of these three genesby nitrate treatment in cpsf30-2 was significantly decreased,while there is almost no significant difference between WTand mutant before nitrate treatment. Accordingly, the foldinductions of these three genes were depressed in the mutant.To verify if these defects are the result of the mutation inCPSF30, the induction of these three genes in two comple-mentation lines, CPSF30-L/cpsf30-2 and CPSF30-S/cpsf30-2,was tested through qPCR analysis. The results showed thatthe expression of these genes was restored to near WT levelsin the CPSF30-L/cpsf30-2 line, but not in the CPSF30-S/cpsf30-2 line when treated with 10 mM nitrate (Fig. 2a), indi-cating that the decreased induction of these three nitrate-responsive genes is caused by the mutation in CPSF30 andthat CPSF30-L plays an important role in nitrate signaling. Inaddition, a significant decrease in induced expression levels of

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Fig. 1 The weak fluorescence phenotype of Mut65 is caused by the mutation in CPSF30 in Arabidopsis. (a) Nitrate induction of NRP-YFP in wild-type(WT) and Mut65 roots. Seedlings (containing the NRP-YFP construct) were grown on nitrate media for 5 d. Light and fluorescent images were capturedwith a fluorescent microscope. (b) Mapping of the gene responsible for the defective response in Mut65. The mutation of Mut65 was mapped to the geneCPSF30 on chromosome 1 showing in the schematic diagram. Amino acid and nucleotide changes found in Mut65 are also shown. (c) Diagram of thestructures of CPSF30. The protein exists in two forms: larger CPSF30 (CPSF30-L) and smaller CPSF30 (CPSF30-S). (d) Complementation test of CPSF30 incpsf30-2. Seedlings of the WT, cpsf30-2, CPSF30-L/cpsf30-2 and CPSF30-S/cpsf30-2were grown on nitrate media for 5 d. Light and fluorescent imageswere captured with a fluorescence microscope. (e) Quantification of root fluorescence. The root fluorescence of the seedlings used in (d) was quantifiedusing ImageJ. Error bars represent SD (n = 50). Different letters indicate statistically significant difference (P < 0.05, t-test).



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the three nitrate-responsive genes was also observed in theRNAi line RNAi-L5-1, similar to that in cpsf30-2 (Fig. 2a).Taken together, these data demonstrate that CPSF30-L func-tions as an important regulatory gene in the primary nitrateresponse.

Thus far it has been found that nitrate regulators may work intwo different ways: modulating nitrate response only whenammonium is present (i.e. without N starvation), which is repre-sented by NRT1.1; and regulating nitrate signaling when

ammonium is both present and absent, which is represented byNLP7 and NRG2 (Castaings et al., 2009; Xu et al., 2016). A pre-vious study showed that the nrt1.1 mutants (chl1-5 and chl1-13)exhibit reduced nitrate-induced expression of the nitrate-inducible genes (NIA1, NiR and NRT2.1), while gene inductionin the mutants can be restored by nitrogen starvation (Wanget al., 2009). YFP fluorescence for chl1-13 was weak on KNO3

medium without nitrogen starvation (Fig. 2b), confirming thatNRT1.1 functions as an important nitrate regulatory gene under

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Fig. 2 Nitrate induction of endogenous genes and fluorescence observation on mutants. (a) Quantitative PCR (qPCR) analysis of nitrate-inducedexpression of NIA1, NiR, and NRT2.1. Wild-type (WT), mutants (cpsf30-2, RNAi line and chl1-13, containing the NRP-YFP construct) andcomplementation lines were grown in hydroponic cultures with 2.5mM ammonium succinate for 7 d and then treated with 10mM KNO3 or KCl as acontrol for 2 h. Root RNA was extracted and determined by qPCR. Error bars represent SD of biological replicates (n = 5) and asterisks indicate a significantdifference (P < 0.05, u-test) compared with the WT. �N, KCl treatment condition; +N, KNO3 treatment condition. (b) Root fluorescence of WT, cpsf30-2,and chl1-13 on medium without nitrogen starvation. Seedlings were grown on ammonium succinate medium for 5 d, and then transferred to KNO3

medium for 16 h. Light and fluorescent images were captured with a fluorescent microscope. (c) qPCR analysis of nitrate-induced expression of NIA1, NiR,and NRT2.1 after nitrogen starvation. Plants were grown and treated as described in the legend to (a), except that plants were transferred to nitrogen-freemedium for 24 h on day 6 and then treated with 10mM KNO3 or KCl for 2 h. Root RNA was extracted and determined by qPCR. Error bars represent theSD of biological replicates (n = 5) and asterisks indicate significant difference (P < 0.05, u-test) compared with the WT. (d) Root fluorescence of WT,cpsf30-2 and chl1-13 on medium with nitrogen starvation. Arabidopsis seedlings were grown on ammonium succinate medium for 3 d, and thentransferred to nitrogen-free medium for 2 d, followed by KNO3 treatment for 16 h. Light and fluorescent images were captured with a fluorescentmicroscope.





these conditions (ammonium present). To test if CPSF30-Lfunctions in the nitrate signaling after nitrogen starvation, thecpsf30-2 mutant was subjected to nitrogen deprivation treatmentfor 24 h after 6 d of growth with ammonium succinate, and thenfollowed by 10 mM KNO3 treatment for 2 h. qPCR analysisshowed that cpsf30-2 exhibited reduced expression levels for thesegenes after nitrate treatment compared with the WT, similar tothe result obtained under the condition without nitrogen depri-vation treatment (Fig. 2c), which is different from nrt1.1mutants. Furthermore, after pretreatment with nitrogen starva-tion, the fluorescence of cpsf30-2 mutant still showed lower YFPfluorescence after nitrate treatment, while strong YFP fluores-cence was restored in chl1-13 (Fig. 2d). These findings indicatethat CPSF30-L functions in nitrate signaling regardless of nitro-gen starvation, belonging to the second type of nitrate regulator.

To investigate whether nitrate regulates the expression ofCPSF30-L, CPSF30-L mRNA levels were measured by qPCR intime-course experiments after 10 mM nitrate treatment. Theresults showed that the expression of CPSF30-L was slightlyinduced by 50% at 1 h after nitrate treatment, while no signifi-cant change was found at other time points tested (Fig. S3a). Theexpression of CPSF30-L was not induced by ammonium treat-ment either (Fig. S3b). However, when the WT plants grown onNH4NO3 for 5 d were subjected to nitrogen starvation, theexpression of CPSF30-L was decreased significantly at 4 h andthen remained relatively stable at lower levels (Fig. S3c). More-over, CPSF30-L showed decreased expression after either nitrateor ammonium deprivation (Figs S3d, 2e). These results suggestthat the expression of CPSF30-L is slightly induced after 1 h ofnitrate treatment and reduced after nitrate and ammonium star-vations, but not regulated by ammonium supply.

We also investigated whether CPSF30-L participates in theresponse to nitrogen starvation or phosphate starvation of theplant. In the time-course experiment, the expression of nitrogenstarvation-induced marker genes NRT2.4 and NRT2.5, which areinvolved in nitrate transport (Kiba et al., 2012; Lezhneva et al.,2014), and CHX17, which functions in potassium transport(Meng et al., 2016), was examined after nitrogen starvation for 2,3, and 4 d from 6-d-old seedlings grown on NH4NO3 medium.The result showed no significant difference for the induced expres-sion of these three genes between the WT and the cpsf30-2mutant(Fig. S4a–c), indicating that CPSF30-L may not be involved innitrate starvation signaling. In addition, two phosphate starva-tion-induced marker genes, PHO1;H1 and PHT1;1 (Wang et al.,2008), were also tested after phosphate starvation for 2, 3, and 4 dand no difference between WT and the mutant was found, imply-ing that CPSF30-L may not function in phosphate starvation sig-naling (Fig. S4d,e). Taken together, these findings suggest thatCPSF30-Lmay be specifically involved in nitrate regulation.

CPSF30-L is mainly expressed in the vascular tissues ofleaves, stems, and flowers, and the protein is localized inthe nucleus

The expression pattern of CPSF30-L was first investigated byqPCR, and the results showed that CPSF30-L is expressed

throughout the plant with strongest expression in the leaves(Fig. 3a). Histochemical analysis using transgenic lines harboringthe GUS gene driven by the CPSF30-L promoter revealed thatCPSF30-L is predominantly expressed in the vascular tissues ofleaves (Fig. 3bi,ii), roots (Fig. 3biii), stems (Fig. 3biv), and flow-ers (Fig. 3biv,v). The expression profile of CPSF30-L suggeststhat it might regulate genes involved in nutrient transport.

It has been reported that the smaller form of CPSF30-L is local-ized in the cytoplasm, and it could be found in the nucleus when itinteracted with other CPSF family members (Rao et al., 2009).To investigate the subcellular localization of the protein, the full-length cDNA of CPSF30-L under the control of 35S promoterwas fused to green fluorescent protein (GFP) and the construct35S:: CPSF30-L-GFP was then introduced into WT plants. Thetransgenic plants showed that the protein is localized in thenucleus (Fig. 3c). When this construct was transiently expressed inN. benthamiana leaf cells, the GFP was also observed in thenucleus (Fig. 3d). Thus, CPSF30-L is a nuclear-localized protein.

Nitrate accumulation in cpsf30-2 is decreased in both rootsand shoots

It has been reported that some nitrate regulators can affect theaccumulation of nitrate within a plant (Castaings et al., 2009;Wang et al., 2009; Xu et al., 2016). To test the physiologicaleffects of CPSF30-L, the nitrate content in plants grown on half-strength MS medium was measured. The results showed that thenitrate accumulation in both roots and shoots of the mutant wassignificantly lower than in the WT (Fig. 4a). This phenotype wasrecovered in the complementation line CPSF30-L/cpsf30-2(Fig. 4a). Thus, CPSF30-L regulates nitrate accumulation inplants. Furthermore, to test if the defective nitrate content incpsf30-2 resulted from altered nitrate absorption, a 15N-labeledexperiment was performed. Nitrate uptake ability in seedlingswas detected and significantly lower levels of nitrates were foundin cpsf30-2 and chl1-13 (as a positive control) mutants than inthe WT. This defect was recovered in the CPSF30-L/cpsf30-2complementation line (Fig. 4b). Taken together, CPSF30-L regu-lates nitrate uptake and accumulation in plants.

The decreased nitrate content phenotype of the mutant led usto investigate the expression of the genes known to be involved innitrate transport and assimilation under half-strength MSmedium. qPCR results showed that the expression of NRT1.1and NRT1.5 in roots was significantly reduced in cpsf30-2 com-pared with the WT (Fig. 4c). As NRT1.1 functions in absorbingnitrate from outside into the roots (Tsay et al., 2007), thedecreased expression of this gene may affect the nitrate uptake.NRT1.5 has been reported to be involved in loading nitrate intothe xylem for root-to-shoot transport (Lin et al., 2008). There-fore, the lower expression of NRT1.5 may result in reduced trans-port from root to shoot. In shoots, NRT1.8, which functions indownloading nitrate from xylem vessel to different tissues (Liet al., 2010), was markedly decreased in cpsf30-2 (Fig. 4d). Inaddition, the expression of NRT1.1 was also found to be signifi-cantly lower in the mutant than in the WT (Fig. 4d). As its func-tion has been reported to be involved in root-to-shoot nitrate



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transport (L�eran et al., 2013), the decreased expression ofNRT1.1 in shoots may account for the reduced nitrate content incpsf30-2 shoots as well. No significant changes were found for theexpression of other NRT1 family genes tested between cpsf30-2and WT (Fig. S5). Furthermore, we also detected the expressionof the CLCa gene which functions in nitrate accumulation as animportant 2NO3

�/1H+ antiporter in plants (Geelen et al., 2000;De Angeli et al., 2006; Monachello et al., 2009). The resultsshowed that there is no significant difference either (Fig. S5).These data suggest that CPSF30-L may modulate the uptake andtranslocation of nitrate, at least partially, through regulating theexpression of NRT1.1 and NRT1.5 in roots, and NRT1.1 andNRT1.8 in shoots.

To test if the altered nitrate accumulation levels in cpsf30-2 arerelated to nitrate assimilation, the expression of several important

genes (NIA1, NIA2, NiR, GLN1.1, and GLN1.3) involved innitrate assimilation was examined. The results showed a higherexpression of NIA, NiR, and GLN1.3 genes in the roots (Fig. 4e)and NiR and GLN1.1 in the shoots of cpsf30-2 than of the WT(Fig. 4f). This enhanced gene expression was reversed in the com-plementation line CPSF30-L/cpsf30-2 (Fig. 4e,f). Previously, thereduced induction of nitrate-responsive genes was a short-termresponse, but the expression of these genes was higher in themutant than in the WT when grown under stable nitrate media,which was a stable long-time response. Most probably the induc-tion of these genes is reduced in the mutant after a short time ofnitrate treatment. But after a long period of nitrate treatment, theplants are adapting to the replete nitrate environment and theexpression of these genes is becoming higher in the mutant thanin the WT. Accordingly, we performed further biochemical

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Fig. 3 Expression pattern of CPSF30-L in different tissues and subcellular localization of CPSF30-L. (a) Quantitative PCR (qPCR) analysis of CPSF30-L geneexpression in different tissues. mRNA was isolated from root, flower, stem, leaf and silique tissues from either 7-d-old seedlings grown on half-strength MSor 45-d-old plants in soil. Error bars represent the SD of biological replicates (n = 5) and asterisks indicate a significant difference (P < 0.05, u- test)compared with the wild-type (WT). (b) Histochemical localization of b-glucuronidase (GUS) activity in ProCPSF30:: GUS plants: (i) cotyledon; (ii) leaf; (iii)root; (iv) stem and flower; (v) flower. (c) Subcellular localization of CPSF30-L protein in Arabidopsis. Confocal laser scanning microscopy pictures andcorresponding brightfield images of Arabidopsis roots were captured from the plants expressing 35Spro:: CPSF30-L-GFP construct (lower panels) or emptyvector as control (upper panels). (d) Subcellular localization of CPSF30-L protein in Nicotiana benthamiana cells. Confocal laser scanning microscopypictures and corresponding brightfield images were captured from N. benthamiana leaf cells transiently expressing 35Spro:: CPSF30-L-GFP construct(lower panels) or empty vector as control (upper panels). Red arrows indicate the nucleus. Bars, 50 lm.





(a) (b)

(c) (d)

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Fig. 4 CPSF30-L regulates nitrate transport and assimilation in Arabidopsis. (a) Nitrate accumulation in both roots and shoots. Seedlings were grownhydroponically on half-strength MS medium for 7 d and then shoots and roots were collected for nitrate content test. (b) Nitrate uptake ability of plantslabeled with 5 mM NH4

15NO3 for 5min. (c, d) Relative expression of NRT1.1 and NRT1.5 in roots (c) and NRT1.1 and NRT1.8 in shoots (d). Wild-type(WT), cpsf30-2, and CPSF30-L/cpsf30-2 plants were grown on half-strength MS medium for 7 d, and then the roots and shoots were collected separatelyfor RNA extraction and determined by quantitative PCR (qPCR). (e, f) Relative expression of several nitrate assimilation genes in roots (e) and shoots (f).Materials, growth condition, sampling, determination, and analysis are the same as (e) and (f) except that specific primers were used to detect theexpression of nitrate assimilation genes. (g, h) Nitrate reductase (NR) activity and amino acid content in cpsf30-2 roots and shoots. The roots and shoots ofthe seedlings grown on half-strength MS medium for 7 d were collected separately for NR activity (g) and amino acid content test (h). Error bars representthe SD of biological replicates (n = 5), and asterisks indicate a significant difference (P < 0.05, u-test) compared with the WT.



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analysis and found that the NR activity in roots was significantlyhigher in cpsf30-2 than in the WT (Fig. 4g). NR proteins are thekey enzymes of nitrate assimilation. The increased NR activitycould result in enhanced amino acid synthesis. Therefore, theamino acid content was measured and, indeed, the result showedsignificantly increased amino acid content in cpsf30-2 roots(Fig. 4h). Although the expression of NiR and GLN1.1 washigher in the mutant, no significant difference was found for NRactivity (Fig. 4g) and amino acid content (Fig. 4h) in shootsbetween the mutant and the WT, which may be a result of thesmall seedlings after 7 d of growth. All these findings demonstratethat CPSF30-L functions as an important regulator in nitrateabsorption, content, and assimilation.

Genetic and molecular evidence suggests that CPSF30-Lworks upstream of NRT1.1 and independently of NLP7 innitrate signaling

As a nitrate sensor, NRT1.1 plays an essential role in nitrate sig-naling (Ho et al., 2009). To better understand the relationshipbetween CPSF30-L and NRT1.1, we investigated their geneexpression in respective mutants. The results revealed that theexpression of NRT1.1 in cpsf30-2 was significantly decreasedunder both KNO3 and NH4NO3 conditions (Fig. 5a), while nosignificant difference was found for the expression of CPSF30-Lin chl1-5 and chl-13 mutants (Fig. 5b) compared with the WT.This indicates that CPSF30-L can regulate the expression ofNRT1.1. To obtain genetic evidence about the relationshipbetween CPSF30-L and NRT1.1, the double mutant cpsf30-2chl1-13 was obtained and characterized. The YFP fluorescencelevels in the double mutant roots on NH4NO3 medium weresimilar to those in chl1-13, but lower than those in cpsf30-2(Fig. 6a,b), suggesting that CPSF30-L may work upstream ofNRT1.1 in nitrate signaling. In addition, the expression ofnitrate-responsive genes was inspected in th edouble mutant. Asshown in Fig. 6(c), the relative expression levels after nitrate treat-ment of these genes in cpsf30-2 chl1-13 were similar to those inthe single mutant chl1-13 and much lower than those in the WT,providing further evidence that CPSF30-L and NRT1.1 functionin the same pathway. Moreover, in the transgenic lines with thefull cDNA of NRT1.1 transformed into cpsf30-2, the phenotypesof weak YFP fluorescence, reduced nitrate content, and inhibitedinduction of nitrate-responsive genes were restored to levels simi-lar/close to those in the WT (Fig. 7a–d). These findings clearlyindicate that CPSF30-L and NRT1.1 work in the same pathwayof nitrate signaling and CPSF30-L functions upstream ofNRT1.1.

Previous studies have shown that CPSF30-L plays animportant role in alternative polyadenylation (Thomas et al.,2012; Liu et al., 2014). Therefore, the regulation of CPSF30-L on the expression of NRT1.1 may be caused by altering thelength of NRT1.1 in the open reading frame (ORF) or 30

untranslated region (UTR). To test this hypothesis, five exonsin the NRT1.1 gene were amplified from the WT, the cpsf30-2 mutant, and the complementation line grown on KNO3,NH4NO3 and NH4Suc medium, respectively. Both the WT

and the mutant showed no difference in band number andsize on electrophoresis gels when using two pairs of primers,indicating that CPSF30-L may not influence the splicing inthe NRT1.1 ORF region (Figs 8a, S6a). Next, we investigatedif 30UTR of NRT1.1 mRNA has different polyadenylatedforms in the cpsf30-2 mutant. The nested PCR technique wasused to amplify the NRT1.1 30UTR in two rounds of PCR.The first round was performed with the NRT1.1-Uf1 (upper)and NRT1.1-Ur (lower) primers, and the second withNRT1.1-Uf2 (upper) and NRT1.1-Ur (lower) primers(Fig. 8b,c). Interestingly, the mutant showed additional bandsin three conditions compared with the WT. All the bandswere cloned and sequenced. The results showed that therewere three common fragments with sizes of 248, 233, and133 bp in the 30UTR of both the WT and cpsf30-2. How-ever, the mutant contained a single band of size 109 bp whichwas almost invisible in the WT (Figs 8d, S6b). In addition,there was a single band of size 176 bp in the WT, but thisfragment was almost invisible in cpsf30-2. The complementa-tion line with the CPSF30-L construct shows the same bandas the WT. However, this different polyadenylation inNRT1.1 30UTR was the same in the mutant under three con-ditions, suggesting that this alternative splicing event was notinfluenced by the presence of nitrate. It has been reportedthat some splicing specificity factors can recognize and bindsome polyadenylation signals upstream of the cleavage site(Chan et al., 2011). Hence we analyzed the 30UTR sequenceof NRT1.1 and found a variant of the polyadenylation signalGATAAA at 16 bp upstream of the cleavage site of form c(Fig. S6b). These results indicate that CPSF30-L could affectalternative polyadenylation in 30UTR of NRT1.1 mRNA.

NLP7 has been reported to be another key nitrate regulator(Castaings et al., 2009; Konishi & Yanagisawa, 2013; Marchiveet al., 2013). To investigate the relationship between CPSF30-Land NLP7, we first examined the expression of NLP7 in cpsf30-2and CPSF30-L in nlp7-4. The results showed no change in bothcases (Fig. S7a,b), indicating that these two genes do not regulateeach other at the transcriptional level. Genetic analysis using sin-gle and double mutants of both genes revealed that under bothNH4NO3 and KNO3 conditions, the YFP fluorescence of cpsf30-2 nlp7-4 was weaker than that of both single mutants (Figs 9a,b,S8a,b), indicating that CPSF30-L and NLP7 may function in dif-ferent pathways in nitrate signaling. In addition, the relativeexpression of the three nitrate-responsive genes tested was signifi-cantly lower in the double mutant than in both single mutants(Fig. 9c), providing further evidence that both genes work inde-pendently in nitrate regulation. Taken these results together, weconclude that CPSF30-L and NLP7 function independently innitrate signaling.

Genome-wide effect of CPSF30 on gene expression

To elucidate the molecular mechanism by which CPSF30 worksin nitrate signaling, we performed a global transcriptome analysison cpsf30-2. Plants were cultured as described in the ‘Transcrip-tome analysis’ subsection. Two biological replicates were tested.





Comparisons of biological replicates showed that their expressionvalues were highly correlated (average R2 = 0.988). In total, 449,778 and 312 clean reads were generated for further analysis afterremoving adaptor sequences and filtering low-quality reads. Foreach sample, c. 85% of reads could be mapped to the Arabidopsis

Information Resource (TAIR) reference genome TAIR10(Table S1b).

To select DEGs in these samples, twofold change and adjustedP-value < 0.05 were used as a cutoff. Our results revealed that theexpression of 633 and 724 genes in the WT and cpsf30-2,

(a) (b)

Fig. 5 The expression of NRT1.1 is reduced in cpsf30-2. (a, b) The expression of NRT1.1 in cpsf30-2 (a) and CPSF30-L in nrt1.1mutants (b). Arabidopsisplants were grown on 10mM KNO3 or NH4NO3 medium for 7 d and whole seedlings were collected for RNA extraction. Primers were designed to amplifythe end of the NRT1.1 coding sequence and used for quantitative PCR (qPCR) analysis. Error bars represent the SD of biological replicates (n = 5), whileasterisks indicate significant difference (P < 0.05, u-test) compared with the wild-type (WT).

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Fig. 6 CPSF30-L and NRT1.1work in the same nitrate signaling pathway. (a) Root fluorescence observation of wild-type (WT), cpsf30-2, chl1-13, andcpsf30-2 chl1-13 double mutant (containing the NRP-YFP construct). Plants were grown on NH4NO3 medium for 5 d. Light and fluorescence images werecaptured with a fluorescence microscope. (b) Quantification of root fluorescence. The root fluorescence of the seedlings as shown in (a) was quantifiedusing ImageJ. Error bars represent SD (n = 50). Different letters indicate statistically significant difference (P < 0.05, t-test). (c) Relative expression of nitrate-induced gene in WT, cpsf30-2, chl1-13 and cpsf30-2 chl1-13mutants. Arabidopsis plant growth conditions, gene expression determination and dataanalysis are the same as in Fig. 2(a). Error bars represent SD (n = 5). Different letters indicate a statistically significant difference (P < 0.05, u-test).



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respectively, was altered significantly after 2 h of nitrate treatment(Fig. 10; Table S2). Among these DEGs, 298 genes (including139 genes up-regulated and 159 genes down-regulated) and 389genes (containing 144 genes up-regulated and 245 genes down-regulated) were specifically differentially expressed in the WTand cpsf30-2 mutant, respectively, while there were also 335 com-mon DEGs (many genes were altered in both WT and mutantafter nitrate treatment, but the fold changes may be different inthese samples) (Fig. 10). We further compared the expressionlevel of these common DEGs, and 202 of the 335 commonDEGs were altered > 20% between the WT and the mutant in

treatment groups (Table S3). GO enrichment was performed forthe combination of 687 specific DEGs and 202 altered commonDEGs. Six enriched GO terms were found to be related to nitro-gen, including response to nitrogen compound, nitrate metabolicprocess, nitrate assimilation, response to nitrate, reactive nitrogenspecies metabolic process, and nitrate transport (Tables 1, S4). Inthese clusters, we found many genes involved in nitrate transportand assimilation, such as NRT1.7, NRT1.8, NRT2.1, NIA1 andNiR. The altered expression of these genes also added anotherpiece of evidence suggesting that CPSF30 regulates nitrate trans-port and assimilation. Moreover, some known nitrate-inducible

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Fig. 7 NRT1.1 can rescue the weak fluorescence phenotype of cpsf30-2 on NH4NO3 medium. (a) Root fluorescence observation in wild-type (WT),cpsf30-2, and NRT1.1/cpsf30-2 plants. Plants (containing the NRP-YFP construct) were grown on NH4NO3 medium for 5 d. Light and fluorescenceimages were captured with a fluorescence microscope. (b) Quantification of root fluorescence. The root fluorescence of the seedlings as shown in (a) wasquantified using ImageJ. Error bars represent SD (n = 50). Different letters indicate statistically significant difference (P < 0.05, t-test). (c) Nitrate content inNRT1.1/cpsf30-2 complementation plants. Plants were grown hydroponically on half-strength MS medium for 7 d and then shoots and roots werecollected for nitrate concentration test. Error bars represent the SD of biological replicates (n = 5), while different letters indicate a significant difference(P < 0.05, u-test) compared with the values of the WT. (d) Relative expression of nitrate-induced gene in WT, cpsf30-2, and NRT1.1/cpsf30-2 plants.Arabidopsis plant growth conditions, gene expression determination, and data analysis are the same as in Fig. 2(a). Error bars represent the SD of biologicalreplicates (n = 5), and asterisks indicate a significant difference (P < 0.05, u-test) compared with the WT.





and regulatory genes, such as NIA1, NRT2.1, and HRS1, werefound in these DEGs (Table 2), which is consistent with ourqPCR result (Fig. 2a). Previously, the transcriptome analysis onanother CPSF30 mutant oxt6 was performed by Delarue’s groupand 2663 DEGs were revealed (Bruggeman et al., 2014). Weanalyzed the 734 DEGs with expression altered more thantwofold and performed GO analysis. Interestingly, a nitrogen-related term (cellular nitrogen compound metabolic process with20 genes) was enriched (Table S1c). The above data providemore evidence that CPSF30 can regulate many nitrogen-relatedgenes and is an important nitrate regulator. In addition, the genesinvolved in the response to chemical, ion and anion transport,and the response to stimulus and stress were also over-represented(Table 1), suggesting that CPSF30 may work as a multifunctionalgene and participate in some other physiological and biochemicalprocesses. Furthermore, we analyzed the 485 DEGs in the nrt1.1mutant compared with the WT (Xu et al., 2016) and found that

123 genes were commonly altered in cpsf30-2 and nrt1.1mutants. These genes were further analyzed by GO analysis and anitrogen-related term ‘response to nitrogen compound’ wasenriched (Table S1d). These data support the conclusion thatCPSF30-L and NRT1.1 work in the same nitrate regulation path-way.

Discussion

Plants have evolved a range of physiological and biochemicalmechanisms to adapt to fluctuating nitrate concentrations in therhizosphere. Nitrate supply is of extreme importance in agricul-ture; however, our understanding of the regulatory mechanismsthat control nitrate uptake and utilization in plants is still incom-plete. In this study, we isolated a new regulatory mutant using aforward genetic screen and mapped the mutation to CPSF30.Our results indicate that the 65 kDa protein encoded by

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Fig. 8 CPSF30-L can affect the NRT1.1mRNA 30 untranslated region (30UTR)alternative polyadenylation. (a) Structure ofthe NRT1.1 gene. (b) Electrophoresis gelscorresponding to the amplified bands ofdifferent 30UTR polyadenylated forms ofNRT1.1 in the wild-type (WT), cpsf30-2complementation line, and NRT1.1-ctransgenic line from KNO3, NH4NO3, andNH4Suc medium. (c) Polyacrylamide gelelectrophoresis gels (PAGE) corresponding tothe amplified bands of different 30UTRpolyadenylated forms in (b). (d) The differentlengths of the 30UTR of NRT1.1mRNApolyadenylated forms in the WT and cpsf30-

2mutant. (a–e) These represent the differentlengths of 30UTR polyadenylated forms.Arabidopsis plants were grown on 10mMKNO3, NH4NO3, or NH4Suc medium for 7 dand whole seedlings were collected for RNAextraction and reverse transcriptionpolymerase chain reaction analysis.



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CPSF30-L is an important regulator in nitrate signaling, as it reg-ulates the expression of genes involved in nitrate uptake, distribu-tion, and assimilation.

The Arabidopsis CPSF30 gene encodes a polyadenylation fac-tor subunit that has both RNA binding and endonuclease activ-ity, is regulated by both calcium and redox signals, and isintimately involved in tolerance to oxidative stress (Delaney et al.,2006; Addepalli & Hunt, 2007; Zhang et al., 2008). In eukary-otes, mRNAs undergo 30 end processing involving an endonucle-olytic cleavage and subsequent polyadenylation that is essentialfor gene expression and regulation (Wahle & R€uegsegger, 1999;Zhao et al., 1999). In mammals, the highly conserved polyadeny-lation signal AAUAAA, providing sequence specificity in bothpre-mRNA cleavage and polyadenylation, can be recognized by aprotein complex that consists of six subunits: CPSF30, CPSF73,CPSF100, CPSF160, Fip1, and Wdr33 (Mandel et al., 2008; Shiet al., 2009). CPSF30 contains five zinc finger motifs and a zincknuckle motif, which could suppress host mRNA 30 processingthrough the interaction with influenza virus NS1A (Nemeroff

(a) (b)

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Fig. 9 NLP7works independently of CPSF30-L in nitrate signaling. (a) Root fluorescence observation of wild-type (WT), cpsf30-2, nlp7-4 and cpsf30-2

nlp7-4 plants (containing the NRP-YFP construct). Plants were grown on NH4NO3 medium for 5 d. Light and fluorescence images were captured with afluorescence microscope. (b) Quantification of root fluorescence. The root fluorescence of the seedlings as shown in Fig. 8(a) was quantified using ImageJ.Error bars represent SD (n = 50). Different letters indicate statistically significant difference (P < 0.05, t-test). (c) Relative expression of nitrate-induced genein WT, cpsf30-2, nlp7-4 and cpsf30-2 nlp7-4 double mutants. Arabidopsis plant growth conditions, gene expression determination, and data analysis arethe same as in Fig. 2(a). Error bars represent the SD (n = 5). Different letters indicate statistically significant difference (P < 0.05, u-test).

Fig. 10 Transcriptome analysis revealed specifically expressed genes in thecpsf30-2mutant. Venn diagram showing the number of the genesregulated by nitrate treatment in the Arabidopsis wild-type (WT) andcpsf30-2mutant.





et al., 1998). In plants, CPSF30 mRNA exists in two forms. Thesmaller one, containing three zinc finger motifs, encodes a28 kDa protein similar to other eukaryotic CPSF30 proteins andhas been shown to participate also in cleavage and polyadenyla-tion site selection on a genomic scale, which can be regulated bycalmodulin (Addepalli & Hunt, 2007; Thomas et al., 2012).Recent studies indicate that CPSF30 plays an important role inplant immune response and the oxt6 mutant also shows defects inlateral root development, fertility, and response to several hor-mones (Hunt, 2014; Liu et al., 2014; Chakrabarti & Hunt,2015). But the function of the gene’s larger form is still unclear.The larger one encodes a 65 kDa protein containing an YTH(YT521 homology) RNA-binding domain in addition to thethree-zinc finger domain. It is the only protein containing bothdomains in Arabidopsis (Li et al., 2014).

Our data indicate that CPSF30-L plays an important role innitrate signaling. An interesting finding from our work is thatonly the 65 kDa protein (CPSF30-L) of CPSF30, containing theYTH domain, could rescue the mutant cpsf30-2 (Figs 1d,e, 2a).Previously, the CPSF30-S was reported to be involved in oxida-tive signaling, as the expression of CPSF30-S, but not CPSF30-L,was induced after oxidative stress by viologen treatment (Zhanget al., 2008). Therefore, it seems that the different forms ofCPSF30 mediate at least two different signaling pathways:CPSF30-L functions in nitrate signaling while CPSF30-S plays arole in oxidative signaling. In eukaryotes, 174 proteins have been

found to contain a YTH domain which consists of 100–150amino acids and is highly conserved (Stoilov et al., 2002). It hasbeen reported that the YTH domain is a novel RNA-bindingdomain that binds to a short, degenerate, and single-strandedRNA sequence motif (Zhang et al., 2010). The human YTHfamily members YTHDF1/2/3 were found to bind N6-methyladenosine-containing RNAs (Wang et al., 2014). InSaccharomyces cerevisiae, Pho92, which contains an YTH domain,could regulate the PHO4 expression through binding to the30UTR in phosphate metabolism (Kang et al., 2014). In plants,microarray data indicated that members of the YTH familymight be involved in growth and development, such as theresponse to stress and hormones (Li et al., 2014). In Arabidopsis,ECT1 and ECT2, belonging to the ECT family containing theYTH domain, are involved in the calcium signal pathway (Oket al., 2005). CPSF30 has been shown to be regulated by calmod-ulin. Interestingly, nitrate signaling also involves calcium signal-ing (Riveras et al., 2015). It is possible that CPSF30 plays a partin the calcium signaling events that mediate nitrate regulation.

Polyadenylation is a key process in mRNA posttranscriptionalregulation in eukaryotes. Alternative polyadenylation playsimportant roles in regulating gene expression by producing dif-ferent lengths of mRNA 30UTR or even diverse protein isoforms.More than 70% of mammalian genes undergo Alternativepolyadenylation (APA), and c. 75% of Arabidopsis annotatedgenes have two or more APA sites (Wu et al., 2011; Derti et al.,2012). In mammals, it is known that APA is a genome-wide reg-ulator of mRNA isoform diversity; for example, some isoformswith shorter 30UTR might lack the cis-elements that microRNAor trans-acting factors could bind compared with the isoformswith longer 30UTR (Proudfoot, 2011; Erson Bensan, 2016).These processes are found to be involved in development,differentiation and disease (Lutz & Moreira, 2011). InChlamydomonas it has been reported that the NZF1 gene, whichencodes a protein with three in-tandem zinc finger motifs similarin structure to CPSF30-L, is involved in nitrate signaling andcontrols the length of the 30 UTR of the nitrate regulatory geneNIT2 (Higuera et al., 2014). The Arabidopsis NLPs are homolo-gous with NIT2 and have been found to modulate the nitrateresponses (Camargo et al., 2007). It will be interesting to deter-mine if Arabidopsis CPSF30-L regulates nitrate signaling by asimilar mechanism as NZF1. Our results showed that the patternof polyadenylation sites in the 30UTR of NRT1.1 mRNA in thecpsf30-2 mutant was changed, suggesting that the CPSF30-L gene

Table 1 Gene ontology (GO) analysis for the Arabidopsis genes specificallyand differentially expressed in the wild-type (WT) and cpsf30-2mutantafter nitrate treatment

Term P-value

Response to stimulus 1.86168E–12Anion transport 1.86168E–12Response to oxygen-containing compound 1.5673E–11Response to chemical 1.5673E–11Inorganic anion transport 2.76188E–11Ion transport 4.19854E–11Response to acid chemical 4.52698E–10Response to stress 1.43072E–09Response to nitrogen compound 4.94169E–08Response to inorganic substance 3.65287E–07Response to external stimulus 2.098E–06Inorganic anion transmembrane transport 2.13157E–06Nitrate metabolic process 2.27731E–06Nitrate assimilation 2.27731E–06Response to nitrate 2.27731E–06Single-organism process 2.43743E–06Reactive nitrogen species metabolic process 3.15197E–06Anaerobic respiration 3.38678E–06Trehalose biosynthetic process 3.72651E–06Nitrate transport 4.76796E–06Trehalose metabolic process 4.76796E–06Response to decreased oxygen concentrations 5.27039E–06Response to organic substance 5.58465E–06Response to oxygen concentrations 5.58465E–06Cellular response to starvation 5.58465E–06Response to starvation 7.95067E–06

Items in bold represent six enriched GO terms related to nitrogen.

Table 2 Some known Arabidopsis nitrate-inducible and regulatory geneswith different induction levels in the wild-type (WT) and cpsf30-2mutant

GeneFold changein WT P-value

Fold changein cpsf30-2 P-value

NIA1 5.93 4.22E–15 4.90 8.44E–24NiR 10.71 9.50E–22 8.66 4.51E–93NRT2.1 4.49 1.12E–05 3.39 5.21E–24HRS1 41.64 3.81E–176 14.84 3.41E–165HHO1 24.83 1.22E–55 17.16 2.42E–83TGA1 3.64 5.17E–27 2.88 4.00E–32



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may affect the polyadenylation processing of NRT1.1 mRNA,resulting in altered expression of NRT1.1. This was also sup-ported by our results showing that the overexpression of NRT1.1in the cpsf30-2 mutant could recover the nitrate content and sig-naling to WT values. Further experiments will be needed todemonstrate that the specific, alternative, polyadenylation sitesfor CPSF30-L are responsible for the regulation of NRT1.1,including transformation of cpsf30-2 mutants, with cDNAencoding the forms c and e of NRT1.1 mRNA.

Previous studies have revealed that nitrate regulators may func-tion in nitrate signaling in at least two different ways: an ammo-nium-dependent manner, as represented by NRT1.1 (Wanget al., 2009; Krouk et al., 2010a); and an ammonium-independent manner, as represented by NRG2 and NLP7 (Xuet al., 2016). Thus far, NRT1.1 is the only gene that has beenfound to regulate nitrate responses when ammonium is present,while losing its nitrate regulation function when ammonium isabsent. In this paper, our results have shown that CPSF30-L reg-ulates nitrate signaling in both the presence and absence ofammonium, similar to NRG2 and NLP7, which is different fromNRT1.1. Moreover, our genetic data indicate that in the presenceof ammonium CPSF30-L works upstream of NRT1.1 in nitratesignaling. It seems that in the absence of ammonium, CPSF30-Lmay regulate the nitrate signaling through gene(s) other thanNRT1.1. Previous ChIP-chip analysis revealed that NLP7 canbind nitrate regulatory genes, including NRT1.1, suggesting thatNLP7 may function upstream of NRT1.1 through direct bindingto NRT1.1 DNA (Marchive et al., 2013). We further tested therelationship between CPSF30-L and NLP7. CPSF30-L andNLP7 could regulate nitrate signaling regardless of the presenceof ammonium, and our molecular and genetic data demonstratedthat CPSF30-L and NLP7 may have additive effects in modulat-ing nitrate response. These results shed new light on the regula-tion of NRT1.1 and identify CPSF30-L as a key component inthe nitrate regulation network.

As sessile organisms, plants have evolved sophisticated mecha-nisms to accommodate limiting and fluctuating environments.Improving the NUE of crops is an important goal for crop pro-duction and sustainable agriculture. The use of nitrate by plantsincludes nitrate uptake, assimilation, and remobilization, andmany genes are involved in these processes, such as NRTs, NR,and GS. Gene variation in different crop species may lead to dif-ferent NUE and grain yield; for example, rice ssp. indica has ahigher nitrate uptake activity than japonica, as a result of a varia-tion in the NRT1.1B (OSNPF6.5) gene (Hu et al., 2015).NRT1.1B-indica contributes to improving the NUE by enhanc-ing nitrate uptake, root-to-shoot transport, and nitrate signaling.Some previously characterized nitrate regulators have beendemonstrated to modulate nitrate uptake and/or assimilation.The nlp7 mutants show higher nitrate content, lower nitratereductase activity, and decreased amino acid content (Castaingset al., 2009). Some genes related to nitrate uptake/assimilationare repressed in LBD37/38/39 overexpression lines, resulting in adecrease in nitrate and total amino acid content (Rubin et al.,2009). NRG2 also modulates the uptake and translocation ofnitrate by regulating the expression of genes involved in nitrate

transport, such as NRT1.1 and NRT1.8 (Xu et al., 2016). In thispaper, our data suggest that CPSF30-L functions as a key playerin nitrate signaling that regulates the expression of genes involvedin nitrate uptake, distribution, and assimilation, and may thus bea useful tool for improving NUE.

Acknowledgements

We thank Prof. Min Ni for discussion of unpublished data andGene denovo Biotechnology (Guangzhou, China) for providingtranscriptome analysis support. This research was supported byNSFC grant (31170230), Taishan Scholar Foundation, andFunds of Shandong ‘Double Tops’ Program to Y.W.

Author contributions

Y.W., R.W., N.M.C., Z.L. and M.Z. designed the research; Z.L.,Y.G., C.W., L.Z., N.X., S.Q. and K-E.C. performed theresearch; Z.L., Y.W. and R.W. analyzed the data; and Y.W.,N.M.C., R.W., Y-F.T. and Z.L. wrote the manuscript.

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Supporting Information

Additional Supporting Information may be found online in theSupporting Information tab for this article:





Fig. S1 CPSF30-L RNAi line target sequence.

Fig. S2 The weak fluorescence phenotype of the CPSF30 RNAiline.

Fig. S3 Expression of CPSF30-L after treatments with differentnitrogen conditions.

Fig. S4 Expression of nitrogen starvation and phosphate starva-tion inducible genes after treatments in the time-course experi-ment.

Fig. S5 Expression of some characterized NRT1 family genes andCLCa in cpsf30-2.

Fig. S6 CPSF30-L cannot affect NRT1.1 exon alternativepolyadenylation.

Fig. S7 A nitrogen-related cluster for genes regulated by bothCPSF30-L and NRT1.1.

Fig. S8 NLP7 works independently with CPSF30-L in nitratesignaling on KNO3 medium.

Table S1 Primers used in this paper, summary of mapped RNAreads, a nitrogen-related cluster for genes differentially expressedinWT and oxt6mutant, and regulated by CPSF30-L andNRT1.1

Table S2 Genes with more than twofold changes in expression inthe WT and cpsf30-2 after nitrate treatment

Table S3 Genes with differentially induced expression levels inthe cpsf30-2 mutant after nitrate treatment

Table S4 Four nitrogen-related clusters for genes differentiallyexpressed in the WT and cpsf30-2 mutant after nitrate induction

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