Article type : Regular Article(Xu et al., 2016), cpsf30-2 (Li et al., 2017) were previously described. Transgenic lines Transgenic lines carrying p 35S :: T5120 were obtained by floral

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nph.16038

This article is protected by copyright. All rights reserved.

PROF. YONG WANG (Orcid ID : 0000-0003-0219-4403)

Article type : Regular Article

The long noncoding RNA T5120 regulates nitrate response and

assimilation in Arabidopsis

Fei Liu1*, Yiran Xu1*, Kexin Chang1, Shuna Li1, Zhiguang Liu2, Shengdong Qi1, Jingbo

Jia1, Min Zhang2, Nigel M. Crawford3, and Yong Wang1,a

1State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural

University, Taian, Shandong 271018, China

2College of Resources and Environment, Shandong Agricultural University, Tai’an,

Shandong 271018, China;

3Section of Cell and Developmental Biology, Division of Biological Science, University of

California at San Diego, La Jolla, California 92093-0116, USA

*These authors contributed equally to this work.

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aAuthor for correspondence:

Yong Wang

Tel: +86 538 8243957

Email: [email protected].

Received: 31 January 2019

Accepted: 20 June 2019

ORCID:

Yong Wang https://orcid.org/0000-0003-0219-4403

Fei Liu https://orcid.org/0000-0001-9922-1281

Jingbo Jia https://orcid.org/0000-0001-6499-5471

Summary

● Long noncoding RNAs (lncRNAs) are crucial regulators in many plant biological

processes. However, it remains unknown whether lncRNAs can respond to nitrate or function

in nitrate regulation.

● We detected 695 lncRNAs, 480 known and 215 novel, in Arabidopsis seedling roots; six

showed altered expression in response to nitrate treatment, among which T5120 showed the

highest induction.

● Overexpression of T5120 in Arabidopsis promoted the response to nitrate, enhanced nitrate

assimilation, and improved biomass and root development. Biochemical and molecular

analyses revealed that NLP7, a master nitrate-regulatory transcription factor, directly bound

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to the nitrate-responsive cis-element (NRE)-like motif of the T5120 promoter and activated

T5120 transcription. In addition, T5120 partially restored the nitrate signaling and

assimilation phenotypes of nlp7 mutant, suggesting that T5120 is involved in NLP7-mediated

nitrate regulation. Interestingly, the expression of T5120 was regulated by the nitrate sensor

NRT1.1. Thus, T5120 is modulated by NLP7 and NRT1.1 to regulate nitrate signaling.

● Our work reveals a new regulatory mechanism in which lncRNA T5120 functions in nitrate

regulation, providing new insights into the nitrate signaling network. Importantly, lncRNA

T5120 can promote nitrate assimilation and plant growth to improve the nitrogen use

efficiency.

Keywords: lncRNAs, nitrate response, nitrate assimilation, T5120, NLP7, Arabidopsis

thaliana

Introduction

Noncoding RNAs (ncRNAs) play vital roles in numerous biological processes in organisms

(Laporte et al., 2007; Rymarquis et al., 2008). One class of ncRNA consists of transcripts

longer than 200 nucleotides with poor protein coding potential, known as long noncoding

RNAs (lncRNAs) (Pang et al., 2006; Ponting et al., 2009). RNA polymerase II synthesizes

most lncRNAs, which are structurally similar to mRNAs, with caps at the 5′ ends and

poly(A) tails at their 3′ ends. Certain lncRNAs are generated by polymerases III, IV, and V

and function as precursors for small interfering RNAs (siRNAs) or as scaffolds in

RNA-directed DNA methylation (Wierzbicki, 2012; Ariel et al., 2014).

Although research into the functions of plant lncRNAs has been limited, the available reports

indicate that lncRNAs, acting through a variety of mechanisms, serve as essential regulators

of various plant biological processes, including gene expression, chromatin remodeling,

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pre-mRNA alternative splicing, and stress responses (Charon et al., 1999; Wierzbicki et al.,

2008; Wilusz et al., 2009; Xin et al., 2011; Bardou et al., 2014). In Arabidopsis thaliana,

numerous lncRNAs have been isolated and characterized. For example, COLD INDUCED

LONG ANTISENSE INTRAGENIC RNA (COOLAIR) and COLD ASSISTED INTRONIC

NONCODING RNA (COLDAIR) transcribed from the FLOWERING LOCUS C (FLC) gene

in the antisense and sense orientations, respectively, are required for epigenetic silencing of

FLC under cold or vernalization conditions (Swiezewski et al., 2009; Heo & Sung, 2011).

The lncRNA IPS1, induced by phosphate starvation, is an endogenous target mimic (eTM) of

miR399, and the binding of IPS1 and miR399 results in the upregulation of target mRNAs of

miR399 (Francozorrilla et al., 2007). The AUXIN REGULATED PROMOTER LOOP

(APOLO) lncRNA is transcribed by RNA polymerases II and V from a gene fragment located

about 5 kb upstream of PINOID (PID), which regulates polar auxin transport (Amor et al.,

2009). The APOLO lncRNA modulates the expression of adjacent gene PID by regulating the

formation of a chromatin loop encompassing the PID promoter (Amor et al., 2009). The

lncRNA HIDDEN TREASURE 1 (HID1) promotes photomorphogenesis by inhibiting the

promoter activity of PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) in continuous red

light (Wang, Y. et al., 2014; Wang, Yuqiu et al., 2014). Alternative splicing competitor long

noncoding RNA (ASCO-lncRNA) is a competitor of target genes of the nuclear speckle

RNA-binding protein (NSR)-containing complex, which modulate mRNA alternative

splicing (Bardou et al., 2014). ELF18-INDUCED LONG NONCODING RNA1 (ELENA1)

affects the expression of PR1 and participates in the plant immune response (Seo et al.,

2017). These results emphasize the importance of lncRNAs and the value of characterizing

more lncRNA functions in plant growth and development as a means to more fully

understand key biological processes.

With the help of next-generation sequencing, a multitude of lncRNAs have been discovered,

but only a few have been characterized (Xin et al., 2011; Banks et al., 2012; Liu et al., 2012;

Meng et al., 2012). To analyze lncRNA functions rapidly and accurately, several

bioinformatic analysis techniques have been developed for predicting the potential target

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genes of lncRNAs, including the prediction of cis-acting, trans-acting, and mimic targets of

miRNAs (Meng et al., 2012; Kang & Liu, 2015). We applied these techniques to our studies

of nitrate regulation.

Nitrate is a crucial signal molecule that modulates plant gene expression, metabolism,

growth, and development (Krouk et al., 2010; Bisseling & Scheres, 2014; Forde, 2014;

Krapp, 2015; Noguero & Lacombe, 2016; O'Brien et al., 2016; Gaudinier et al., 2018; Zhao,

Lufei et al., 2018). Plants have evolved sophisticated mechanisms that allow them to adapt to

fluctuations in nitrate availability. Nitrate responses are generally divided into two phases.

The primary nitrate response is defined as the short-term effects of nitrate exposure (Medici

& Krouk, 2014; Zhao, Lufei et al., 2018). Transcriptional analysis and experimental

validation indicate that the expression of more than 1000 genes including NRT2.1, NIA1, and

NIR is altered after nitrate treatment (Wang et al., 2003; Scheible et al., 2004; Wang,

Rongchen et al., 2004; Xu et al., 2016; Li et al., 2017). Recently, some genes containing

NRT1.1, NLP6/7, LBD37/38/39, SPL9, TGA1/4, CIPK8/23, NRG2, CPSF30-L, and FIP1

have been identified as important regulators in primary nitrate response (Alvarez et al., 2014;

Medici & Krouk, 2014; Xu et al., 2016; Wang, C et al., 2018; Wang, YY et al., 2018; Fredes

et al., 2019). The long-term nitrate response is defined as nitrate’s effects on aspects of longer

term plant growth and development including seed dormancy, root system architecture, the

circadian system, and stomatal movement (Roenneberg & Rehman, 1996; Forde, 2002; Guo

et al., 2003; Walchliu et al., 2006; Wilkinson et al., 2007; Bisseling & Scheres, 2014; Forde,

2014; Guan et al., 2014; O'Brien et al., 2016). Some microRNAs (miRNAs) have been

reported to participate in nitrate regulation (Miin-Feng et al., 2006; Vidal et al., 2010). For

example, miR393 can be induced by nitrate and regulates primary and lateral root growth in

response to nitrate (Vidal et al., 2010). Moreover, the levels of miR167 are repressed under

nitrogen treatment, resulting in the accumulation of the auxin response factor ARF8, and the

miRNA/ARF8 regulatory module has an essential role in modulating lateral root growth in

response to nitrogen (Miin-Feng et al., 2006). As for lncRNAs, it remains largely unclear if

they function in nitrate regulation.

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The nodule inception protein-like (NIN) protein NLP7 is a master regulator involved in the

primary nitrate response and in long-distance regulation of nitrate signaling (Castaings et al.,

2009). NLP7 belongs to the NLP family, whose members are characterized by two conserved

domains, the RWP-RK and PB1 domains. NLP7 is a transcription factor, and its RWP-RK

domain is responsible for DNA binding (Schauser et al., 2005). A chip-based chromatin

immunoprecipitation (ChIP-chip) assay showed that NLP7 directly binds to the

nitrate-responsive cis-element (NRE) of many nitrogen-related genes (Konishi &

Yanagisawa, 2013; Marchive et al., 2013). In addition, the conserved Ser205 residue in NLP7

can be phosphorylated by CPK10/30/32, leading to its nuclear retention in the presence of

nitrate (Marchive et al., 2013; Liu et al., 2017). Interestingly, NLP7 is also involved in

long-term effects; for example, it’s overexpression can promote root development under

nitrate-rich conditions (Yu et al., 2016). Recently, two homologous genes (ZmNLP6 and

ZmNLP8) in the maize (Zea mays) NLP family have been characterized as encoding proteins

that play important roles in enhancing nitrate assimilation, root growth, and nitrogen use

efficiency (NUE) when ectopically expressed in Arabidopsis (Cao et al., 2017).

During the last several years, RNA-seq and small RNA sequencing (sRNA-seq) have been

used to identify key factors in nitrate signaling, and there are several reports describing

genome-wide identification of lncRNA responses to nitrogen deficiency or nitrogen treatment

in Populus and maize (Vidal, E. A. et al., 2013; Alvarez et al., 2014; Chen et al., 2016; Lv et

al., 2016). As yet, no lncRNAs have been reported to respond to nitrate or to be involved in

nitrate regulation in Arabidopsis. In this work, we identified 695 lncRNAs containing 480

previously known and 215 novel lncRNAs in Arabidopsis seedling roots using RNA-seq

technology. Differentially expressed genes (DEG) analysis and qRT-PCR validation

confirmed that the transcript levels of six of these lncRNAs were significantly altered after

nitrate treatment. Among these, the lncRNA showing the highest nitrate induction, T5120,

was induced by nitrate directly. Moreover, T5120 regulated nitrate response, nitrate

assimilation, and plant growth. Further investigation revealed that T5120 was regulated by

NLP7 through its direct binding to the T5120 promoter. T5120 partially restored the

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phenotypes of the nlp7 mutant, indicating that T5120 functions in the nitrate regulatory

pathway mediated by NLP7. Moreover, expression of T5120 was regulated by NRT1.1,

suggesting that T5120 is modulated by NRT1.1 to regulate nitrate regulation. These results

indicate that the lncRNA T5120 plays an important role in nitrate regulation and will provide

a fundamental base for future exploration of the functions of lncRNAs in nitrate regulation.

Materials and Methods

Plant materials and growth conditions

Col-0 was used as the wild-type (WT) ecotype of Arabidopsis thaliana, and homozygous

transgenic seeds carrying a nitrate-responsive yellow fluorescent protein reporter construct

(NRP-YFP), were used as WT for the observation of fluorescence signals (Wang et al.,

2009). The three nlp7 mutant lines (nlp7-1, nlp7-2, and nlp7-4 containing NRP-YFP)

(Castaings et al., 2009; Xu et al., 2016) and the transgenic lines (pNLP7::NLP7-GFP/nlp7-1

and p35S::NLP7-GFP/nlp7-1) (Marchive et al., 2013) were previously described. The mutant

lines chl1-5 (Ho et al., 2009), cipk8-1 (salk_139697) (Hu et al., 2009), nrg2-2 (salk_079096)

(Xu et al., 2016), cpsf30-2 (Li et al., 2017) were previously described. Transgenic lines

carrying p35S::T5120 were obtained by floral dipping (Clough & Bent, 2010) of the WT and

nlp7-4 mutant. All primers used here are listed in Table S1.

To perform the RNA-seq assay and measure the nitrate induction of lncRNAs, seedlings were

sterilized, plated in aseptic solution containing 2.5 mM ammonium succinate as the sole

nitrogen source for 7 d, and then treated either with 10 mM KNO3 or with 10 mM KCl as a

control for 2 h. To examine the expression profiles of lncRNAs, plants were grown on

half-strength Murashige and Skoog (MS) medium for 7 d or in soil under long-day (LD;

light/dark, 16 h/8 h) conditions for 40 d. For the transient dual-luciferase reporter assay,

plants were grown in soil under short-day (SD; light/dark, 8 h/16 h) conditions for 30 d.

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RNA-seq and bioinformatics analysis

Four sets of samples, comprising two treatments and two controls (WT-KNO3 and WT-KCl),

with two biological replicates each were subjected to RNA-seq. Total RNAs eliminating

ribosome RNA were used for strand-specific library construction and sequenced using an

Illumina HiSeqTM2500 platform by Gene Denovo Biotechnology (Guangzhou, China).

Reads obtained from sequencing were filtered to remove adapters and low-quality reads using

FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and rRNA was

removed by mapping reads to an rRNA database using Bowtie 2 (Langmead & Salzberg,

2012). The clean reads from each sample were mapped to the reference genome TAIR 10

(http://www.arabidopsis.org) with TopHat 2 (version 2.0.3.12) (Kim et al., 2013) and then

assembled with Cufflinks (Trapnell et al., 2016). All transcripts were merged into a final

comprehensive set of transcripts for further analysis using Cuffmerge.

Identification of lncRNAs

All reconstructed transcripts were aligned to the reference genome and were divided into 12

categories using Cuffcompare. The transcripts aligned with annotated lncRNAs were defined

as known lncRNAs. Novel transcripts were screened using two criteria: the length of the

transcript was longer than 200 bp and the number of exons was more than 1 (Derrien et al.,

2012; Zhan et al., 2016). To obtain novel lncRNAs, two software packages, CNCI (version 2)

and CPC (http://cpc.cbi.pku.edu.cn/), were used to evaluate the protein-coding potential of

the novel transcripts using default parameters (Kong et al., 2007; Sun et al., 2013). The

intersection of the two sets of results was defined as novel lncRNAs. The sequences of the

novel lncRNAs verified by qPCR in this paper were submitted to the GenBank with the

accession numbers MN096324 (TCONS_00005120), MN096325 (TCONS_00010478), and

MN096326 (TCONS_00016715).

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Identification of nitrate-responsive lncRNAs

The software RESM was used to quantify transcript abundances (Li & Dewey, 2011). The

transcript expression level was normalized using the fragments per kilobase of transcript per

million mapped reads (FPKM) method. The differentially expressed lncRNAs were analyzed

using the edgeR package (http://www.r-project.org). LncRNAs with a fold change of ≥ 2 and

a false discovery rate (FDR) of ≤ 0.05 were defined as nitrate-responsive lncRNAs.

qRT-PCR assay

Plant samples frozen in liquid nitrogen were ground using tissue crushers (Retsch), and then

RNA was extracted using an Ultrapure RNA Kit (CWBIO). Reverse transcription was

performed with an lnRcute lncRNA First-Strand cDNA Synthesis Kit (with gDNase)

(Tiangen Biotech). Real-time quantitative polymerase chain reactions were performed with

LightCycler®96 (Roche) using UltraSYBR Mixture (CWBIO). Tubulin (AT5G62690) was

used as an internal reference gene for normalizing the relative expression of lncRNAs. All

primers used here are listed in Table S1.

Nitrate content, 15NO3- uptake, nitrate reductase activity, and amino acid content assays

Nitrate content was measured using the hydrazine reduction method as described by Cao et

al. (2017). The assay of 15NO3- uptake was performed as described previously (Wang &

Tsay, 2011). Determination of nitrate reductase (NR) activity was done by sulfanilamide

colorimetric analysis based on a previously published description (Zhao, L. et al., 2018). The

amino acid content was tested using the ninhydrin colorimetric method according to a

previous description (Rosen, 1957).

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Yeast one-hybrid assay

The full-length NLP7 cDNA was cloned into the pJG4 vector and then AD-NLP7

recombination was performed. To generate a construct with the promoter of T5120 driving

the LacZ reporter gene, two complementary primers containing putative NRE cis-elements in

the T5120 promoter were synthesized, annealed, and then digested with KpnI and SalI and

ligated into the pLacZ-2u vector. The recombined constructs were cotransferred into yeast

competent cells and cultivated on SD/-Trp/-Ura medium with X-gal. Yeast one-hybrid

analysis was performed according to a previous description (Lin et al., 2007; Li et al., 2011).

All primers used here are listed in Table S1.

ChIP-qPCR analysis

ChIP-quantitative PCR (ChIP-qPCR) assays were performed according to a protocol

described previously with minor modification (Saleh et al., 2008; Zhonghai et al., 2013).

Briefly, seedlings carrying pNLP7::NLP7-GFP were grown on half-strength MS medium for

7 d, collected, fixed in 1% formaldehyde for 30 min in a vacuum and then neutralized with

0.125 M Gly for 5 min in a vacuum. The samples were washed twice with distilled deionized

H2O, frozen with liquid nitrogen, and ground to powder. Extraction buffer was used to lyse

cells, and nuclei were isolated. Nuclei were crushed in nuclei lysis buffer and then sonicated.

The chromatin supernatant was divided into three aliquots. One sample was used as input for

a positive control, a second was combined with protein G-agarose beads and anti-GFP

antibodies, and the third was combined with beads but without antibodies as a negative

control to exclude nonspecific precipitation. The samples (except the input sample) were

incubated at 4� for 8 h and then the beads were successively washed with low-salt, high-salt,

LiCl, and TE buffers, twice per buffer. Elution and reverse cross-linking was performed with

elution buffer and 5 M NaCl at 65°C. DNA was purified as previously described (Zhonghai

et al., 2013). Primers used in ChIP-qPCR are listed in Table S1. The enrichment of DNA

fragments was determined by qPCR.

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Transient dual-luciferase reporter assay

A 2158-bp T5120 promoter DNA fragment was amplified by PCR and then inserted into the

pGreen II 0800 vector containing the firefly luciferase (LUC) reporter gene. The construct

carrying the full-length cDNA of NLP7 was used as the effector. Protoplasts were isolated

according to Sheen (http://genetics.mgh.harvard.edu/sheenweb) as described previously (Li et

al., 2005). Protoplasts were cotransfected with pT5120::LUC and p35S::NLP7 or

pT5120::LUC and empty vector and incubated under dark conditions for 16 h. The firefly

luciferase/renilla luciferase (LUC/REN) ratio was monitored following the protocol of the

TransDetect Double-Luciferase Reporter Assay Kit (TransGen Biotech) using a luminometer

(Promega) as previously described (Guo et al., 2017). All primers used here are listed in

Table S1.

Results

Genome-wide identification of lncRNAs in Arabidopsis roots

To explore the lncRNAs involved in nitrate regulation and identify the lncRNAs responding

to nitrate, we performed high-throughput strand-specific RNA-seq of transcripts, which has

recently emerged as a powerful tool for recognizing novel transcripts (Liu et al., 2013; Vidal,

Elena A et al., 2013; Di et al., 2014). The samples used for sequencing were from

Arabidopsis Col-0 seedlings grown for 7 d on medium containing NH4+ as sole nitrogen

source and then treated for 2 h either with 10 mM KNO3 or with 10 mM KCl as control. For

each library, we obtained 80-110 million 125-bp-long raw reads and removed low-quality

reads, adapters, and reads that mapped to rRNA to obtain high-quality clean data. We aligned

the clean reads to the Arabidopsis Information Resource TAIR 10

(http://www.arabidopsis.org) with TopHat 2 and assembled the clean reads that matched to

the reference genome using Cufflinks and Cuffmerge (Table S2, Fig. S1). Comparison of the

assembled transcripts to the annotated mRNA and lncRNA sequences identified 480 known

lncRNAs (Table S3). We subjected novel transcripts to further strict filtering based on the

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following criteria: 1) the lengths of the transcripts were >200 bp, 2) the novel transcripts

contained at least two exons, 3) the transcripts had low protein-coding potential as predicted

by CPC and CNCI software (Fig. S2). After filtering, we identified 215 transcripts as novel

lncRNAs (Table S4). Thus, we identified 695 lncRNAs, of which 480 were known and 215

were novel.

Identification of nitrate-responsive lncRNAs

To identify the lncRNAs responding to nitrate, we defined lncRNAs with a fold change of

≥1.5 and P value of ≤0.05 between the WT-KNO3 and WT-KCl groups as being differentially

expressed. According to these criteria, 35 lncRNAs were differentially expressed after nitrate

treatment (Table S5): 23 known lncRNAs, of which 13 were upregulated and 10 were

downregulated, and 12 novel lncRNAs, of which 6 were upregulated and 6 were

downregulated (Fig. S3).

To identify the lncRNAs whose expression was most significantly changed by nitrate

treatment, we strengthened the filtering criteria by increasing the required fold change to

more than 2.0 and reducing the false discovery rate (FDR) to lower than 0.01. Using these

criteria, we identified eight lncRNAs (Table 1), four of which were novel. We then used

qPCR to validate the differences in the expression of these lncRNAs after nitrate treatment

compared to that without nitrate treatment. We were unable to design primers to test the

expression level of TCONS_00017071 because of its sequence overlap with AT2G44798 and

AT2G44800. The qPCR results showed that the expression of six of the seven lncRNAs was

increased after nitrate treatment; the exception, TCONS_00010478, showed no change in

expression in the qPCR results but reduced expression by RNA-seq (Fig. 1a and S4).

Furthermore, the expression trends of nitrate-related lncRNAs from the RNA-seq and qPCR

data were closely correlated, based on their fold change after nitrate treatment (R2 = 0.81, P <

0.05) (Fig. 1b). These results indicate that these six lncRNAs validated by qPCR are indeed

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responsive to nitrate. In addition, considering the potential interaction between lncRNAs and

their adjacent genes, we analyzed the neighboring genes within a 10 kb distance either

upstream or downstream from the lncRNAs (Table S6). The target analyses provide a

direction for further investigation of the functions of these lncRNAs.

Expression profiles of nitrate-responsive lncRNAs in different tissues

To better understand the functions of the nitrate-responsive lncRNAs, we examined their

expression in different tissues using qPCR. The transcript abundances of these lncRNAs

exhibited diverse patterns (Fig. 2). The highest expression of AT1G13448 was in mature

rosette leaves, while that of AT1G67105 occurred in cauline leaves (Fig. 2a, b). AT2G35637

was preferentially expressed in flowers, although its expression was very low in all organs

tested (Fig. 2c). The expression of AT3G17185 was higher in flowers and siliques than in

other organs (Fig. 2d). TCONS_00005120 had the lowest expression of all the tested

lncRNAs in all organs (Fig. 2e), but it also showed the strongest nitrate induction (Fig. 1a).

Finally, TCONS_00016715 was specifically expressed in flowers while showing little

expression in other organs (Fig. 2f). These results suggest possible directions for further

study of these lncRNA functions.

TCONS_00005120 expression is regulated in a time- and concentration-dependent

manner by nitrate but not its reduction products

Since the expression of TCONS_00005120 (hereafter called T5120) was markedly induced by

nitrate treatment (Fig. 1a), we further analyzed its expression pattern in response to nitrate.

First, we performed nitrate treatment time-course experiments to measure T5120 expression

over time by growing WT seedlings on ammonium succinate medium for 7 d and then

exposing them to 10 mM KNO3 for up to 8 h. T5120 expression in whole seedlings was

induced rapidly by nitrate (Fig. 3a). The T5120 transcript level was increased after nitrate

treatment at 0.25 h, peaked at 1 h, and fell back to basal levels after 8 h.

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To determine whether the induction of T5120 was affected by nitrate concentrations, we

examined T5120 expression in WT seedlings planted on medium with ammonium as the sole

nitrogen source and then treated with different concentrations of nitrate for 1 h. qRT-PCR

analysis showed that T5120 induction was more pronounced after treatment with higher

compared to lower nitrate concentrations (Fig. 3b), indicating that the induction of T5120 by

nitrate is concentration dependent.

Nitrate has been reported to act as a signaling molecule that directly regulates the expression

of some genes (Wang, Rongchen et al., 2004; Wang et al., 2007). To determine whether

T5120 is such a gene, we analyzed the nitrate response of T5120 in a NR-null (nia1 nia2)

mutant (Wang, R. et al., 2004) and found that T5120 was also induced by nitrate in the

NR-null mutant (Fig. 3c), indicating that T5120 is directly induced by nitrate and does not

require production of downstream reduction products of nitrate. These results demonstrate

that T5120 is specifically responsive to nitrate and may play important roles in the nitrate

response.

T5120 modulates the nitrate response and nitrate assimilation

To investigate whether T5120 functions in nitrate regulation, we constructed transgenic lines

overexpressing T5120 in the WT background (Fig. S5) and then examined the expression of

three endogenous nitrate-responsive genes: NIA1, NIR, and NRT2.1. All three genes were

much more strongly induced in the T5120-overexpressing (T5120-OE) lines than in the WT

after KNO3 treatment (Fig. 4a), indicating that T5120 participates in the primary nitrate

response. To verify if T5120 affects nitrate metabolism, we first measured the accumulation

of nitrate in T5120-OE lines grown on half-strength MS medium and found that it was

significantly lower than that in the WT (Fig. 4b).

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Furthermore, to test whether the reduced nitrate content in the T5120-OE lines resulted from

altered nitrate uptake, we determined the 15NO3- uptake by submerging WT and T5120-OE

plants in 10 mM NH415NO3 for 30 min. The results showed that 15NO3

- uptake did not differ

significantly between WT and T5120-OE lines (Fig. 4c). To confirm these results, we

measured the expression of several genes involved in nitrate absorption (NRT1.1, NRT1.2,

NRT2.1, and NRT2.2). We found no significant difference in the expression of any of these

genes between T5120-OE and WT (Fig. S6). Thus, T5120 overexpression does not affect

nitrate absorption. Subsequently, we examined the effects of T5120 on nitrate assimilation

and found that the NR activity and amino acid content in T5120-OE lines were significantly

higher than those in the WT (Fig. 4d, e), indicating that T5120 regulates nitrate assimilation.

To investigate the mechanism underlying the higher nitrate assimilation in T5120-OE plants,

we next investigated the expression of several genes involved in nitrate assimilation. The

expression levels of four of those genes, NIA1, NIA2, NIR, and GLN1.1, were considerably

higher in T5120-OE lines than in the WT (Fig. 4f). These results confirmed that T5120

regulates nitrate response and nitrate assimilation.

NLP7 binds to and activates the promoter of T5120

To further characterize the function of T5120, we analyzed the sequence of the T5120

promoter and identified an NRE-like motif located at positions -464 to -1 from the

transcription start site (Fig. 5a). The NRE is a conserved sequence, first identified in the

promoter of the Arabidopsis gene NIR1, and acts as an essential cis-element in nitrate

response (Konishi & Yanagisawa, 2010). Furthermore, NLP transcription factors are

NRE-binding proteins that directly modulate the expression of some nitrate-related genes

(Castaings et al., 2009; Konishi & Yanagisawa, 2013; Sato et al., 2016). To investigate

whether NLP7 can directly bind to the NRE-like motif of the T5120 promoter, we performed

a yeast one-hybrid (Y1H) assay. First, we created two constructs for a fusion protein of NLP7

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with the activation domain of a galactose-regulated upstream promoter element (GAL4) and

a fragment containing the NRE-like element of the T5120 promoter fused with the LacZ

reporter gene. NLP7 bound to the NRE-like motif of the T5120 promoter and activated the

LacZ reporter in vitro (Fig. 5b).

Second, to confirm the binding of NLP7 to the NRE-like element of the T5120 promoter, we

performed ChIP-qPCR analysis with anti-GFP antibodies on samples from the

pNLP7::NLP7-GFP/nlp7-1 transgenic plants treated with KNO3 or KCl. After nitrate

treatment, the enriched abundance of the T5120-NRE fragment was much higher in the

sample with antibody than in the sample without antibody, while the enriched abundances of

ACTIN12 and T5120-UN fragments, as negative controls, showed no significant differences

between the two samples. ChIP-qPCR results did not show enrichment of the T5120-NRE

fragment when plants were treated with KCl, suggesting that NLP7 cannot bind to the T5120

promoter in the absence of nitrate (Fig. 5c). These results indicate that NLP7 binds to the

NRE cis-element of the T5120 promoter in vivo in the presence of nitrate.

Finally, to confirm the transcriptional activity of NLP7 on the T5120 promoter, we performed

a dual-luciferase reporter assay. We generated a construct containing the firefly luciferase

gene (LUC) driven by the T5120 promoter and the Renilla luciferase gene (REN) driven by

the cauliflower mosaic virus (CaMV) 35S promoter as the reporter and cotransfected this into

protoplasts either with an effector plasmid for expression of NLP7 or with an empty vector as

a control. The resulting fluorescence data showed that NLP7 activated the T5120 promoter

(Fig. 5d). These findings indicate that the activity of the T5120 promoter is modulated by

NLP7 through its direct binding to the promoter.

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NLP7 regulates T5120 expression

Given that NLP7 can bind to the T5120 promoter, we wondered whether it could modulate

T5120 expression. We used the WT, nlp7, and NLP7/nlp7-1 complementation lines with

different transcriptional levels of NLP7 to investigate the expression of T5120 (Fig. S7).

T5120 expression was significantly reduced in the nlp7 mutants and increased in the

NLP7/nlp7-1 complementation lines (Fig. 5e), indicating that NLP7 does regulate T5120

expression.

To test whether NLP7 is required for the induction of T5120 in response to nitrate, we

examined T5120 induction in plants treated with 10 mM KNO3. Induction of T5120 was

dramatically inhibited in nlp7 mutants and was restored to WT levels in the

pNLP7::NLP7-GFP/nlp7 line, while the expression of T5120 was over-induced in the

p35S::NLP7-GFP/nlp7 line (Fig. 5f). These results indicate that NLP7 is essential for the

response of T5120 to nitrate.

T5120 partially restores nitrate signaling and assimilation of nlp7-4

NLP7 has been reported to be a key factor regulating nitrate signaling and assimilation

(Castaings et al., 2009). Considering that T5120 expression is modulated by NLP7 (Fig. 5e,

f), we next investigated whether T5120 functions in NLP7-mediated nitrate regulation. We

overexpressed T5120 in the nlp7-4 mutant and found that the resulting transgenic lines

showed higher expression of T5120 than occurred in either the WT or the nlp7-4 mutant (Fig.

S8). When the plants were grown on nitrate medium, the root YFP fluorescence driven by a

nitrate-inducible promoter (NRP) in the T5120/nlp7-4 lines was stronger than that of the

nlp7-4 mutant and weaker than that of the WT (Fig. 6a, b). Furthermore, we determined the

expression of three genes involved in nitrate assimilation, NIA1, NIA2, and NIR, in

T5120/nlp7-4 plants and found that it was greater than that in the nlp7-4 mutant, but still

lower than that in the WT (Fig. 6c). Moreover, the nitrate reductase activity of T5120/nlp7-4

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lines was higher than that of the nlp7-4 mutant, being partially restored to WT levels (Fig.

6d). The above results indicate that T5120 plays an important role in NLP7-mediated nitrate

signaling.

NRT1.1 regulates the expression of T5120

To investigate the relationship of T5120 with other known nitrate-regulatory genes in

addition to NLP7, we measured the expression of NRT1.1, CIPK8, NRG2, and CPSF30-L.

The results exhibited no significant difference between T5120-OE lines and WT (Fig. 7a),

suggesting that T5120 may not regulate the expression of these genes. We then examined the

expression of T5120 in different nitrate-regulatory mutants including chl1-5 (nrt1.1 mutant),

cipk8-1, nrg2-2, and cpsf30-2. The expression of T5120 was significantly decreased in chl1-5

mutant, but unchanged in other tested mutants (Fig. 7b), indicating that the expression of

T5120 can be regulated by NRT1.1.

T5120 improves plant growth under nitrate-limiting and -sufficient conditions

As T5120 enhances nitrate assimilation, it seemed useful to test whether T5120 could affect

plant growth. Therefore, we grew the plants on the media with deficient and sufficient nitrate

(0.2 and 10 mM KNO3) for 10 d and then measured plant fresh weight, primary root length,

and lateral root number. The fresh weight of the T5120-OE lines was significantly higher

than that of the WT under both conditions (Fig. 8a, b). Moreover, the length of the primary

root and the number of lateral roots of T5120-OE lines were also significantly increased than

those of the WT under both nitrate conditions (Fig. 8c-e). These findings demonstrate that

T5120 can improve plant growth under both nitrate-limiting and -sufficient conditions.

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Discussion

LncRNAs have been reported to regulate various plant biological processes, such as

vernalization, photomorphogenesis, and phosphate homeostasis (Francozorrilla et al., 2007;

Swiezewski et al., 2009; Heo & Sung, 2011; Wang, Y. et al., 2014). However, it is still

unknown whether lncRNAs function in nitrate signaling and metabolism. In this study, we

identified six lncRNAs that respond to nitrate through RNA-seq and qPCR assays (Table 1,

Fig. 1a); this is the first attempt, to our knowledge, to focus on the functions of lncRNAs in

nitrate responses in Arabidopsis. One of these nitrate-responsive lncRNAs, AT3G17185

(TAS3a), has been reported to control lateral root growth by inhibiting the expression of

auxin-responsive factors (ARF2, ARF3, and ARF4) (Elena et al., 2010). As nitrate can

integrate with auxin signaling to affect the root system architecture (RSA) (Miin-Feng et al.,

2006; Bielach, 2010; Elena et al., 2010), the lncRNA AT3G17185 may play a role in root

growth through nitrate-integrated auxin signaling. Here, we investigated the function of the

lncRNA T5120 and demonstrated that T5120 was strongly induced by nitrate and played

important roles in nitrate signaling, nitrate assimilation, and plant growth.

To elucidate the role of T5120 in nitrate metabolism, it was important to dissect how it

functions in nitrate uptake, assimilation, and accumulation. We found that T5120-OE lines

showed decreased nitrate accumulation (Fig. 4b) and significantly increased nitrate reductase

activity, amino acid content, and expression of nitrate-assimilatory genes (Fig. 4d-f), while

nitrate uptake was unchanged (Fig. 4c). Thus, the reduced nitrate content in T5120-OE lines

was likely due to enhanced nitrate assimilation. The 15NO3- tracer assays showed no effect of

T5120 on nitrate uptake and expression studies exhibited no significant differences in the

expression of nitrate transporter genes (NRT1.1, NRT1.2, NRT2.1, and NRT2.2) between

T5120-OE and WT plants (Figs. 4c and S6). Therefore, T5120 does not appear to regulate

nitrate absorption. The reason that the induction of NRT2.1 by nitrate is increased (Fig. 4a)

and nitrate uptake is unchanged (Fig. 4c) in T5120-OE is most probably because that the

induction of NRT2.1 in T5120-OE plants is transient: higher than that in the WT after a

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short-time nitrate treatment but returning to WT levels after a long-time treatment, as the

plants are adapting to the sufficient nitrate environment.

The NLP proteins have been proposed to serve as central transcription factors in nitrate

regulation. Several pathways have been characterized for NLP7’s regulation of nitrate

signaling. NLP7 directly binds to some nitrate-related genes, including NIR, NRT2.1, and

NRT2.2, controlling their transcription levels and then modulating nitrate transport and

assimilation (Castaings et al., 2009; Konishi & Yanagisawa, 2013; Marchive et al., 2013;

Guan et al., 2017). Moreover, NLP7 also regulates the nitrate signaling by directly binding to

the promoter and controlling the expression of NIGT1, which encodes the GARP-type

transcriptional repressor 1 proteins and affects the expression of some target genes involved

in nitrate regulation (Maeda et al., 2018). Interestingly, our study reveals that NLP7 works

upstream of T5120 to regulate the nitrate signaling. Previous reports showed that the

localization of NLP7 is regulated by nitrate. When nitrate is present, the Ser205 in NLP7 is

phosphorylated by CPK10/30/32, the phosphorylated NLP7 protein accumulates in the

nucleus, then NLP7 can bind to the NRE elements to regulate the expression of target genes

(Konishi & Yanagisawa, 2013; Marchive et al., 2013; Liu et al., 2017). Considering that the

T5120 promoter contains a NRE-like motif (Fig. 5a), we looked for possible binding of NLP7

to T5120’s promoter using yeast one-hybrid and ChIP-qPCR assays and confirmed that this

occurs (Fig. 5b, c). The phenotypes observed using ChIP-qPCR revealed that NLP7 binds to

the T5120 promoter after but not before nitrate treatment, which may result from the nuclear

retention of NLP7 protein in the presence of nitrate (Fig. 5c). From a ChIP-chip analysis,

NLP7 had been reported to bind 851 genes (Marchive et al., 2013), but whether it bound

lncRNA DNAs was unknown. Our results showed that NLP7 binds the lncRNA T5120 gene

and regulates its expression and works upstream of T5120 in nitrate regulation. We also

found that another central regulator is involved. NRT1.1 has been reported to act as a nitrate

sensor and to regulate the expression of many nitrate-responsive genes (Walch-Liu & Forde,

2008; Ho et al., 2009; Wang et al., 2009; Krouk & Gojon, 2011; Bouguyon et al., 2015).

Interestingly, we found that the expression of T5120 was significantly reduced in the chl1-5

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mutant (Fig. 7), indicating that NRT1.1 regulates T5120 expression. Therefore, these data

demonstrate that T5120 is modulated by NLP7 and NTRT1.1 to regulate nitrate signaling.

With the rapid development of RNA-seq technology, an increasing number of lncRNAs have

been discovered in plants (Liu et al., 2012; Liu et al., 2013; Wang et al., 2015); however,

only a few have been characterized (Heo & Lee, 2015; Shafiq et al., 2016). In those few, the

regulatory mechanisms of lncRNAs are complex. For example, some (such as IPS1) serve as

the target mimics of miRNAs that alter the miRNAs’ effects on their target genes

(Francozorrilla et al., 2007). Others (such as HID1) directly bind to the promoters of target

genes and regulate their expression (Wang, Y. et al., 2014). Still others (like ASCO-lncRNA)

interact with proteins to regulate the expression of target genes (Bardou et al., 2014). Our

study reveals a new function for lncRNAs: regulation of nitrate responses and assimilation by

T5120. Given that T5120 is positioned adjacent to NIA1 on the chromosome and regulates its

expression (Table S6 and Fig. 4a, f), it is possible that T5120 may modulate the expression of

NIA1 by affecting chromatin remodeling or epigenetic modification, in a fashion reminiscent

of the regulatory actions of the lncRNAs APOLO and COOLAIR on their adjacent genes

(Amor et al., 2009; Swiezewski et al., 2009). More work is required to test this possibility

and to unravel the function of T5120 more fully.

Improving the nitrogen use efficiency (NUE) of crops is of great importance for crop

production and sustainable agriculture. Generally, NUE depends on the nitrogen uptake

efficiency (NUpE) and nitrogen assimilation efficiency (NUtE) (Xu et al., 2012). Some

nitrate-regulatory genes have been reported to be involved in nitrate utilization: for example,

NRT1, NRT2, and GS (Martin et al., 2006; Hu et al., 2015; Fan et al., 2016). Our results show

that T5120 enhances nitrate assimilation, increases seedling biomass, and improves primary

and lateral root growth (Fig. 4d-e and Fig. 8). Therefore, T5120 may be of great value for

improving NUE of plants.

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Acknowledgements

We would like to thank Dr. Anne Krapp for the pNLP7::NLP7-GFP/nlp7-1 and

p35S::NLP7-GFP/nlp7-1 seeds, Dr. Yi-Fang Tsay for cipk8-1 seeds, and Dr. Kang Yan for

discussion of unpublished data. This work was supported by the National Natural Science

Foundation of China (grant number 31670247), the Taishan Scholar Foundation, Funds of

Shandong “Double Tops” Program (grant number SYL2017YSTD01), and National Science

and Technology Major Project (2016ZX08003005-009) to Y.W.

Author contributions

Y. W., N. M. C., F. L., and Y. X. planned and designed the research. F. L., Y. X., K. C., S.

L., and Z. L. performed experiments. Y. W., F. L., Y. X., S. Q., and J. J. analyzed the data. Y.

W., N. M. C., M. Z., and F. L. wrote the manuscript. All authors read and approved the final

manuscript. FL and YX contributed equally to this work.

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Figure legends

Fig. 1 Identification of lncRNAs responsive to nitrate in Arabidopsis seedling roots. (a)

Validation of differentially expressed lncRNAs from RNA-seq data by qPCR. LncRNA

expression determined by qPCR of samples from wild-type (WT) seedlings grown on 2.5

mM ammonium succinate medium for 7 d and then treated for 2 h with either 10 mM KNO3

or 10 mM KCl (as a control). Error bars represent ± standard deviation of biological

replicates (n = 4). Asterisks indicate significant differences (P < 0.05, u-test). (b) Nitrate

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induction levels of lncRNAs determined by qPCR and RNA-seq were closely correlated. FC,

fold change.

Fig. 2 Expression patterns of six nitrate-responsive lncRNAs in different organs. Tissues

were obtained at different developmental stages from Arabidopsis seedlings grown on

half-strength MS medium for 7 d (seedling, seedling root, and seedling leaf) and 40-day-old

plants grown in soil (rosette leaf, cauline leaf, stem, flower, and silique) and expression levels

were measured by qPCR. Transcript levels of six lncRNAs (a-f) were normalized to Tubulin

expression levels. Error bars represent ± standard deviation of biological replicates (n = 4).

Significant differences (P < 0.05, u-test) are indicated by different letters.

Fig. 3 T5120 is induced by nitrate as a signal molecule. (a, b) Relative expression of T5120

was tested by qPCR under different treatments. Arabidopsis seedlings (WT) were grown on

medium with 2.5 mM ammonium succinate for 7 d and then treated with 10 mM KNO3 for

the indicated periods (a) or with different KNO3 concentrations for 1 h (b). Error bars

represent ± standard deviation of four biological replicates. Significant differences (P < 0.05,

u-test) are indicated by different letters. (c) Relative expression of T5120 in WT and NR-null

mutant (nia1 nia2) seedlings grown on medium with 2.5 mM ammonium succinate for 7 d

and then treated with 10 mM KNO3 or 10 mM KCl as a control for 1 h. Error bars represent ±

standard deviation of biological replicates (n = 4). Asterisks indicate significant differences

(P < 0.05, u-test).

Fig. 4 T5120 regulates nitrate response and assimilation. (a) Expression of

nitrate-inducible genes were determined by qPCR in the Arabidopsis roots of wild-type (WT)

and T5120-OE seedlings plated on 2.5 mM ammonium succinate medium for 7 d and then

treated with 10 mM KNO3 or KCl for 2 h. Error bars represent ± standard deviation of

biological replicates (n = 4). Asterisks indicate significant differences (P < 0.05, u-test). (b)

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Nitrate contents of WT and T5120-OE plants. 7-d-old seedlings grown on half-strength MS

medium were tested for nitrate content. Error bars represent ± standard deviation of four

biological replicates. Asterisks indicated significant differences (P < 0.05, u-test). FW, fresh

weight. (c) 15NO3- uptake in WT and T5120-OE plants grown in liquid medium with 5 mM

NH4NO3 for 10 d and then treated with 10 mM NH415NO3 for 30 min. Error bars represent ±

standard deviation of biological replicates (n = 3). DW, dry weight. (d, e) Nitrate reductase

activity (d) and amino acid content (e) in WT and T5120-OE plants. The same conditions for

plant growth were used as in (b). Error bars represent ± standard deviation of four biological

replicates. Asterisks indicate significant differences (P < 0.05, u-test). (f) Expression of

nitrate-assimilatory genes were determined by qRT-PCR in WT and T5120-OE seedlings

grown on half-strength MS medium for 7 d. Error bars represent ± standard deviation of four

biological replicates. Asterisks indicate significant difference (P < 0.05, u-test).

Fig. 5 NLP7 regulates the expression of T5120 through direct binding to the T5120

promoter. (a) Alignment of the NRE-like motif in the T5120 promoter and conserved NRE

sequence. (b) Yeast one-hybrid assays showed the direct binding of NLP7 to the NRE-like

element in the T5120 promoter in vitro. AD, activation domain of GAL4; AD-NLP7,

AD-fusion protein of NLP7. (c) ChIP-qPCR assay showed that NLP7 bound to the NRE-like

fragment in T5120 promoter in vivo. pNLP7::NLP7-GFP/nlp7-1 transgenic seedlings were

grown on 2.5 mM ammonium succinate medium for 10 d, treated with 10 mM KNO3 or KCl

(as a control) for 2 h, and then harvested for the ChIP assay using anti-GFP antibodies.

anti-GFP(+), with antibodies; anti-GFP(-), without antibodies (a negative control).

T5120-NRE, fragment containing the NRE motif; ACTIN12 and T5120-UN, fragments

cannot be bound by NLP7 and were used as controls. Error bars represent ± standard

deviation of biological replicates (n = 3). (d) Regulation of T5120 transcription activity

through the binding of NLP7 to the T5120 promoter was determined using a transient

dual-luciferase reporter assay. The constructs pT5120::LUC and p35S::NLP7 or an empty

vector were transiently cotransformed into Arabidopsis protoplasts and then the LUC/REN

ratio was monitored after culturing the protoplasts under dark conditions for 16 h. Error bars

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represent ± standard deviation of biological replicates (n = 4). (e, f) Expression and nitrate

induction of T5120 were reduced in nlp7 mutants. RNA was extracted from different

seedlings containing wild-type (WT), nlp7-1, and NLP7/nlp7-1 complementation lines

(pNLP7::NLP7-GFP and p35S::NLP7-GFP) grown on half-strength MS medium to test the

T5120 expression (e). WT, nlp7-1, and NLP7/nlp7-1 were grown on 2.5 mM ammonium

succinate for 7 d and then treated with 10 mM KNO3 or KCl as a control for 2 h. RNA was

obtained to measure the induction of T5120 after nitrate treatment (f). Error bars represent ±

standard deviation of biological replicates (n = 4). Different letters indicate statistically

significant differences (P < 0.05, u-test).

Fig. 6 T5120 partially restores the fluorescence phenotype, expression of

nitrate-assimilation genes, and NR activity of the nlp7-4 mutant. (a) YFP fluorescence in

the Arabidopsis roots of wild-type (WT), nlp7-4, and T5120/nlp7-4 transgenic plants grown

on medium containing 10 mM NH4NO3 for 5 d. (b) Quantification of root fluorescence of the

seedlings from (a) using ImageJ. Error bars represent ± standard deviation of biological

replicates (n = 60). Statistically significant differences (P < 0.05, u-test) are indicated by

different letters. (c) Relative expression of nitrate-assimilatory genes were determined by

qPCR in WT, nlp7-4, and T5120/nlp7-4 plants grown on half-strength MS medium for 7 d.

Error bars represent ± standard deviation of four biological replicates. Statistically significant

differences (P < 0.05, u-test) are indicated by different letters. (d) NR activity in the WT,

nlp7-4, and T5120/nlp7-4. Plant growth conditions were as in (c). Error bars represent ±

standard deviation of four biological replicates. Significant differences (P < 0.05, u-test) are

indicated by different letters.

Fig. 7 The expression of T5120 was reduced in the chl1-5 mutant. Arabidopsis plants

were grown on half-strength MS medium medium for 7 d and whole seedlings were collected

for qPCR assay. (a) Expression of NRT1.1, CIPK8, NRG2, and CPSF30-L in T5120-OE

lines. (b) Expression of T5120 in chl1-5, cipk8-1, nrg2-2, and cpsf30-2 mutants. Error bars

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represent the ± standard deviation of three biological replicates; asterisks indicate significant

difference (P < 0.05, u-test) compared to the wild-type (WT).

Fig. 8 T5120 improves plant growth under both nitrate-limiting and -sufficient

conditions. (a) Arabidopsis seedlings grown horizontally on medium with 0.2 or 10 mM

KNO3 for 10 d, respectively. Bars, 1 cm. (b) Fresh weight of the plants from (a). Error bars

represent ± standard deviation of three biological replicates, and each replicate contains 40

plants. Asterisks indicate significant difference (P < 0.05, u-test). (c) Seedlings grown

vertically on medium with 0.2 or 10 mM KNO3 for 10 d, respectively. Bars, 1 cm. OE-1,

T5120-OE-1 transgenic line; OE-2, T5120-OE-2 line. (d) Primary root length and (e) lateral

root number of the plants from (c). Error bars represent ± standard deviation of three

biological replicates, and each replicate contains 30 plants. Asterisks indicate significant

difference (P < 0.05, u-test).

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Table 1 LncRNAs differentially expressed in the roots of Arabidopsis treated with

WT-KNO3 and WT-KCl.

ID FC Log2(FC) FDR significant

AT1G13448.1 13.958217 3.803043 1.88E-15 yes

AT1G67105.1 2.365455 1.242118 0.000104 yes

AT2G35637.1 5.378378 2.427171 0.038406 yes

AT3G17185.1 2.304253 1.2043 2.11E-05 yes

TCONS_00005120 2150 11.0701 0.001274 yes

TCONS_00010478 0.396946 -1.33298 0.023209 yes

TCONS_00016715 2.061224 1.043502 0.009454 yes

TCONS_00017071 15.81484 3.983205 5.08E-14 yes

ID, lncRNA ID; FC, fold change of WT-KNO3 versus WT-KCl; FDR, false discovery rate.

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Supporting Information

Additional supporting information may be found in the online version of this article.

Fig. S1 Diagram of the pipeline for RNA-seq bioinformatics analysis.

Fig. S2 Detailed flowchart for identification of lncRNAs and nitrate-responsive lncRNAs in

Arabidopsis roots.

Fig. S3 Differentially expressed lncRNAs with fold change ≥ 1.5 and P value ≤ 0.05 in

WT-KNO3 and WT-KCl.

Fig. S4 Validation of differentially expressed lncRNAs from RNA-seq data by qPCR.

Fig. S5 Expression of T5120 in WT and in WT transgenic lines overexpressing T5120.

Fig. S6 The expression of nitrate transporter genes in WT and T5120-OE plants.

Fig. S7 Expression of NLP7 in the WT, nlp7 mutants, and nlp7-1 mutant transgenic lines

expressing NLP7.

Fig. S8 Relative expression of T5120 in the WT, nlp7-4 mutant, and nlp7-4 mutant transgenic

lines expressing T5120.

Table S1 Primers used in this research.

Table S2 Summary of read counts.

Table S3 Identification of known lncRNAs in Arabidopsis roots.

Table S4 Identification of novel lncRNAs in Arabidopsis roots.

Table S5 LncRNAs differentially expressed with fold change ≥ 1.5 and P value ≤ 0.05 in

WT-KNO3 versus WT-KCl.

Table S6 Target gene prediction of six nitrate-responsive lncRNAs in cis-acting.

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Documents

Article type : Regular Article(Xu et al., 2016), cpsf30-2 (Li et al., 2017) were previously described. Transgenic lines Transgenic lines carrying p 35S :: T5120 were obtained by floral