AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip

Abstract

Nitrogen and phosphorus are among the most widely used fertilizers worldwide. Nitrate (NO₃^- ) and phosphate (PO₄^3- ) are also signalling molecules whose respective transduction pathways are being intensively studied. However, plants are continuously challenged with combined nutritional deficiencies, yet very little is known about how these signalling pathways are integrated. Here we report the identification of a highly NO₃^- -inducible NRT1.1-controlled GARP transcription factor, HRS1, document its genome-wide transcriptional targets, and validate its cis-regulatory elements. We demonstrate that this transcription factor and a close homologue repress the primary root growth in response to P deficiency conditions, but only when NO₃^- is present. This system defines a molecular logic gate integrating P and N signals. We propose that NO₃^- and P signalling converge via double transcriptional and post-transcriptional control of the same protein, HRS1.

ARTICLE

Received 16 Jun 2014 | Accepted 12 Jan 2015 | Published 27 Feb 2015

Anna Medici1, Amy Marshall-Colon2,w, Elsa Ronzier1,w, Wojciech Szponarski1, Rongchen Wang3, Alain Gojon1, Nigel M. Crawford3, Sandrine Ruffel1, Gloria M. Coruzzi2 & Gabriel Krouk1

Nitrogen and phosphorus are among the most widely used fertilizers worldwide. Nitrate (NO3 ) and phosphate (PO43 ) are also signalling molecules whose respective transduction pathways are being intensively studied. However, plants are continuously challenged with combined nutritional deciencies, yet very little is known about how these signalling pathways are integrated. Here we report the identication of a highly NO3 -inducible

NRT1.1-controlled GARP transcription factor, HRS1, document its genome-wide transcriptional targets, and validate its cis-regulatory elements. We demonstrate that this transcription factor and a close homologue repress the primary root growth in response to P deciency conditions, but only when NO3 is present. This system denes a molecular logic gate integrating P and N signals. We propose that NO3 and P signalling converge via double transcriptional and post-transcriptional control of the same protein, HRS1.

DOI: 10.1038/ncomms7274

AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip

1 Biochimie et Physiologie Molculaire des Plantes, Institut Claude Grignon, UMR5004 CNRS/INRA/Supagro-M/UM2, Place Viala, F-34060 Montpellier cedex 2, France. 2 Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York 10003, USA. 3 Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California 920930116, USA. w Present addresses:

Department of Plant Biology, University of Illinois, 1201W Gregory Drive, 193 ERML MC-051, Urbana, Illinois, USA (A.M.-C.); Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York, New York 14642, USA (E.R.). Correspondence and requests for materials should be addressed to G.K. (email: mailto:[email protected]

Web End [email protected] ).

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As sessile organisms, plants have evolved a myriad of adaptive mechanisms to cope with nutritional limitations in their environment. In particular, plants adapt their

root development differently according to nutritional cues1. Nitrate (NO3 ) and phosphate (PO43 ), two major phytomacronutrients, are also well known signalling molecules shaping root development through partly dened molecular pathways26. For the inorganic phosphate (Pi) response, prd7, lpr1, lpr2 (ref. 8), pdr2 (refs 9,10), phr1 (refs 11,12) and siz1 (ref. 13) mutations have been found to affect the primary root growth. Concerning NO3 , its effect is more related to lateral root growth, with the implication of several molecular actors (thoroughly reviewed in refs 4,14), and has been shown to counteract the effect of glutamate on the primary root growth15.

Despite this knowledge, a mechanism by which a plant integrates the presence or the absence of combinations of such key nutritional molecules is still largely unknown. Recently, the NLA (nitrogen limitation adaptation) and PHO2 proteins (two ubiquitin ligases) were found to control PO43 transporter trafcking (PHT1 family), which results in a nitrogen-dependent

PO43 accumulation in leaves1619. Despite these rst hints into the molecular connections between P and N nutrition, nothing has so far been revealed concerning the mechanisms by which NO3 affects PHO2 or NLA activities.

The root tip is known to be at the forefront of Pi sensing8. It is also the territory of expression of the recently identied nitrate sensor NRT1.1 (refs 20,21). Thus, it represents the perfect place for such nutritional signalling interactions. The present work reports such missing mechanism. Here we show that HRS1 and HHO1 are two early NO3 -regulated transcription factors (TFs).

We document genome-wide HRS1 direct targets and demonstrate that hrs1;hho1 double mutant is involved in the primary root growth repression in response to a combination of N and P signals. We report a potential mechanism to explain this phenotype as HRS1 is under a dual transcriptional and post-transcriptional control. Finally, an in planta genome-wide investigation provides potential signalling pathways under HRS1 and HHO1 inuence.

ResultsIdentication of two NO3 -regulated TFs. A set of early nitrate-regulated gene clusters have been previously identied in a genome-wide investigation22. Among these, At1g13300 was one of the most rapidly and strongly upregulated TFs (with a signicant response recorded within 6 min after treatment). The early and marked induction of At1g13300 and several genes involved in the nitrate uptake and assimilation have been conrmed in an independent set of experiments (Fig. 1). In roots, NO3 provision triggers the induction of the two sentinel gene transcripts (NIR and NRT2.1) after 10 and 30 min respectively. This validates the conditions of the N treatment (Fig. 1a). As predicted by the previous experiments, At1g13300 accumulation was rapidly and strongly upregulated (10-fold within 10 min) compared with the KCl mock treatment (Fig. 1a). These observations are consistent with the previous reports corresponding to genome-wide or specic reverse transcription quantitative (qPCR) investigations6,2325.

The At1g13300 gene encodes a myb-related TF belonging to the GARP (GOLDEN2, ARR-B, Psr1) family; a homologue to NIGT1 identied in rice26. The GOLDEN2-like subgroup is composed of 40 protein sequences (AGRIS, http://arabidopsis.med.ohio-state.edu/

Web End =http://arabidopsis. http://arabidopsis.med.ohio-state.edu/

Web End =med.ohio-state.edu/ ). It encompasses proteins involved in various processes ranging from the control of chloroplast and leaf development (GLKs27 and KANADIs28) to nutritional reprogramming in plants (PHR1 and PHL1 (ref. 12)). A phylogenetic tree has been built out of the 40 G2-like protein

sequences (Supplementary Fig. 1, see Methods). It shows that At1g13300 belongs to a small group of seven proteins sharing high sequence similarity to each other, but quite well separated from the rest of the tree (Fig. 1c; Supplementary Fig. 1). This subgroup within the larger GARP family has already been reported on its own29. We thus kept the proposed nomenclature and extended the relationships between the other members of the GARP family. At1g13300 is named HRS1 (hypersensitive to low Pi-elicited primary root shortening 1)29. HRS1 is a paralogue with its closest neighbour, At3g25790, which has been named HHO1 (HRS1 homologue 1)29. It is noteworthy that these two close homologues, HRS1 and HHO1, have recently been identied among the 2,594 gene pairs that were dened in an effort to isolate redundant duplicated genes30. Interestingly, HHO1 showed a similar pattern of expression to HRS1 with a strong upregulation (450-fold) within the rst 20 min of nitrate treatment (Fig. 1a), both being mainly expressed in roots (Fig. 1d). Furthermore, HRS1 and HHO1 messenger RNA (mRNA) levels in roots are dependent on nitrate concentration in the media. Wild Type seedlings constantly grown for 14 days on media containing increasing NO3 concentrations presented a gradual transcriptional response of HRS1 and HHO1 (Fig. 1b).

Interestingly, despite the fact that both genes are upregulated by NO3 provision, the increasing KNO3 concentration has opposite effects on their steady-state expression levels: HRS1 is positively regulated and HHO1 is negatively affected (Fig. 1b). In conclusion, HRS1 and HHO1 are two root-specic, NO3 -controlled TFs (found to be responsive in the nitrate reductase-null mutant in previous genome-wide investigations31). A meta-analysis6 also demonstrated that HRS1 and HHO1 are under the control of NRT1.1 protein activity. Indeed, their nitrate induction is strongly and robustly affected in chl1 mutants (defective in the NRT1.1 gene) in two independent transcriptomic data sets issued from two independent laboratories25,32. We also performed analysis in our conditions and indeed recorded defects in nitrate responsiveness in an independent chl1 deletion allele of NRT1.1 (chl1-12; Fig. 1e). In the chl1-12 mutant, the 24-fold induction (after 30 min of NO3 treatment) of HRS1 expression is totally abolished and the HHO1 strong induction (B100-fold) is reduced to a third. Finally, HRS1 and HHO1 are also under the inuence of NLP genes (NIN-like TFs) implicated in N-signalling33, and are found to be bound (assayed by chromatin immunoprecipitation sequencing) by NLP7 (ref. 34).

Collectively, these results demonstrate that the TF paralogues, HRS1 and HHO1 are each strongly NO3 controlled (for example, are among the most, if not the most, robustly NO3 -regulated genes in many data sets35) and positioned downstream of the early regulators NRT1.1/CHL1 and NLP7-6 activity.

Identication of HRS1 direct targets. At the outset of this study nothing was known about the molecular mechanism downstream of At1g13300 (HRS1). We thus decided to investigate the genome-wide effect of this TF using a transient assay system for TF perturbation that can uncover direct targets only36. Indeed, we have shown for other TFs (ABI3 and bZIP1) that using this system to uncover the direct regulated targets of a TF can give very important clues about its functional role in planta36,37. We thus speculated that we could retrieve the functional in planta activity of At1g13300 by studying the set of its direct targets.

To search for such genes that are directly regulated by At1g13300, we used the TARGET (Transient Assay Reporting Genome-wide Effects of Transcription factors) approach described by Bargmann et al.36 Briey, protoplasts are isolated from roots of 10-day-old Arabidopsis seedlings and transformed with the plasmid pBeaconRFP_GR-HRS1, expressing a

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551 regulated probes

mRNA level

4

5 NIR

4 KCl KNO3

NRT2.1

3

4 HRS1 Induced

Repressed

3

2

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3

mRNA level

2

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1

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0 0 10 20 30

Log 10(pval)

4.03.53.02.52.01.5

4 2 2 4

0

1

0

Min Min6 HRS1 0.8

1.0 HHO1

2.5

0

0.05

0.5

1

2.5

mRNA level

2.0

HHO1

mRNA level

4

1.5

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0.6

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0.2

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AT1G49560

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AT4G37180

45

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AT2G03500

HRS1

HHO4

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98

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66

AT1G13300

HHO1

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AT3G25790

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76

AT1G25550

HHO2

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45

AT1G68670

0.00

HRS1 HHO1

Repressed

2.5 NRT2.1

4

6 HRS1 2.0

HHO1

2.0

2.5 KCl KNO3

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1.5

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Col chl1-12

0

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0.0

Col chl1-12

Figure 1 | HRS1 and HHO1 are two Arabidopsis transcription factors highly induced by NO3 downstream the nitrate sensor activity. Identication of HRS1 direct targets. (a) Dynamic nitrate response of mRNA for NIR, NRT2.1 (NO3 responsive sentinels), HRS1 (At1g13300) and HHO1 (At3g25790) in roots of 14-day-old seedlings treated with 1 mM KNO3 or 1 mM KCl (as mock treatment), values are meanss.e.m. (n 4). (b) Steady-state level of HRS1

and HHO1 mRNA in roots of plants grown for 14 days on solid media containing 0.5 mM KH2PO4 and different KNO3 concentrations, values are meanss.e.m. (n 4). (c) A subfamily of the G2-like transcription factors phylogenetic tree built by ClustalW alignment and maximum likelihood method,

values are bootstraps based on 500 replicates (see Supplementary Fig. 1). (d) HRS1 and HHO1 mRNA levels in roots and leaves of WT plants grown for 14 days on basal MS media containing 0.5 mM KH2PO4 and 2.5 mM KNO3, values are meanss.e.m. (n 3). (e) HRS1 and HHO1 nitrate induction is

affected in roots of chl1-12 mutant mutated in NRT1.1. WT and chl1-12 14-day-old seedlings were treated for 30 min with 1 mM KNO3 or 1 mM KCl (as mock treatment). All transcript levels were quantied by reverse transcription qPCR and normalized to two housekeeping genes (ACT and CLA), values are meanss.e.m. (n 4). (f) Volcano plot of HRS1 direct regulated targets identied by TARGET (transient assay reporting genome-wide effects of

transcription factors) procedure36. (g) Semantic gene enrichment analysis results showing overrepresented terms in the list of the HRS1 direct up- and downregulated genes.

translational fusion (N-ter) between HRS1 and the rat glucocorticoid receptor (GR) under the control of the pCaMV35S promoter. Protoplasts are treated with (i) dexamethasone (DEX; triggers GRTF fusion entrance in the nucleus) and (ii) cycloheximide (CHX; translation inhibitor that prevents activation of indirect targets)36. Because of the possibility that HRS1 acts as a part of a NO3 -regulated protein complex (as it is itself NO3 regulated, see above), we kept NO3

present during the whole TARGET procedure. After uorescent-activated cell sorting selection (based on red uorescent protein signal provided by an independent cassette in the plasmid) of transformed protoplasts, total RNA was isolated from GRHRS1 protoplasts and used for the transcriptomic analysis. The statistical analysis (see Methods) of transcriptome results identied 551 gene probes whose expression was affected by GR-HRS1 nuclear import. The corresponding genes were classied as up- and downregulated HRS1 direct target genes (Fig. 1f; Supplementary Data 1).

To search for the functions that are globally affected by HRS1 activity, we performed two kinds of analysis. First, we determined over-represented gene ontology (GO) categories using the VirtualPlant and agriGO platforms38,39. Second, we

developed our own algorithm that search for over-represented terms in a set of TAIR v10 gene description (see Methods). Both approaches yielded very similar conclusions: At1g13300 (HRS1) collectively induces genes related to phosphate and cell division (including the terms meristematic activity, cyclin and ribosomal proteins). On the other hand, in the list of HRS1 downregulated genes, the most frequent terms were heat shock and the most represented functions were linked to response to stresses (Fig. 1g; Supplementary Data 1; Supplementary Fig. 6). This genome-wide investigation of the At1g13300 HRS1 TF direct targets brought us to be interested in the interaction between NO3 signalling (that controls transcriptional activation of this gene) and P nutrition that seems to be one of the functions under its inuence29. Taken together with the fact that HRS1 belongs to the same TF family as the very well characterized PHR1 gene11,12, known to be central in the control of P starvation response, we decided to employ a reverse genetic approach to understand the role of HRS1 in the control of NO3 and Pi signalling interactions.

HRS1 and HHO1 repress primary root growth. The hrs1-1 mutant was obtained from ABRC seeds stock center, and the

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T-DNA insertion and the absence of HRS1 full-length transcripts were conrmed (Supplementary Fig. 2). First, we tested the growth of the hrs1-1 mutant on different media containing P

(0.5 mM KH2PO4) or P combined with different NO3 con

centrations (0, 0.05, 0.5, 1 and 2.5 mM). The results did not lead to any reliable/robust phenotype (Supplementary Fig. 3a), as previously reported29. We thus hypothesized that this could be due to functional redundancy of the two NO3 -induced close relatives HRS1 and HHO1 (Fig. 1c). We thus tested the double mutant hrs1-1;hho1-1 (see molecular characterization in Supplementary Fig. 2) on the same P/N varying conditions. As previously shown8, the primary root growth of WT plants was impaired on P conditions. Interestingly, however, the primary

root growth of the double hrs1;hho1 mutant was strongly insensitive to the P conditions (Fig. 2a,b). This insensitivity

is also manifested on transfer experiments (Supplementary Fig. 3b,c). The fact that the hsr1;hho1 double mutant is resistant to P conditions complements previous observations

reporting that the overexpression of HRS1 indeed confers hypersensitivity to low P conditions29. More importantly, NO3

is required for the manifestation of the hrs1;hho1 double mutant phenotype. Indeed, the phenotype of the hrs1;hho1 double mutant is lost in plants grown on N/ P conditions. More

precisely, the presence of at least 0.05 mM NO3 in the media was necessary for the appearance of the root phenotype in response to

Pi-depleted conditions (Fig. 2c). These results suggest that the HRS1 and HHO1 proteins act as repressors of primary root development specically when PO43 is absent and NO3 is present in the media. It is thus tempting to propose that the well-known primary root growth repression in response to low P conditions is an active process that is inuenced by (i) NO3 and(ii) the NO3 -regulated TFs HRS1 and HHO1.

HRS1 and HHO1 are expressed in elongating root cell nuclei. To further investigate the in planta roles of the HRS1 and HHO1 proteins, we constructed native promoter gene green uorescent protein (GFP) lines for these two TFs. Epiuorescence and confocal imaging of transgenic pHRS1:HRS1:GFP and pHHO1:HHO1:GFP plants indicate that the two proteins are expressed and localized in the nucleus of epidermal and cortex cells in elon-gating roots (Fig. 2d; Supplementary Fig. 4). HRS1 and HHO1 expression is found in the transition domain of the root apical meristem and in the elongation zone (Fig. 2d). The expression of HRS1 was also visible in root hair cells (Supplementary Fig. 4). The localization of these two proteins supports the idea that

0.5 mM KH2PO4

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Primary root length (cm) Primary root length (cm)

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Transition

Division

HRS1:GFP

0.5 mM KH2PO4

Elongation

0.5 mM KNO3

Figure 2 | HRS1 and HHO1 are two nuclear factors expressed in meristematic elongating cells repressing root growth in response to combination of P and N availability. (a) Primary root growth measurement over time of Col and hrs1-1;hho1-1 seedlings grown on P/N combinations. In P/ N

conditions, the WT and mutant slopes overlap. (b) Nine-day-old seedlings grown on 0.5 mM of NO3 and PO43 under different conditions (00.5 mM added). (c) Effect of different nitrate concentrations in the media on the primary root growth of hrs1-1;hho1-1 9 day-old seedlings. Values represent the meanss.e.m. (n 30). Asterisks indicate signicant differences from WT plants (*Po0.05; ***Po0.001; Students t-test). (d) Epiuorescent and

confocal imaging of pHRS1:HRS1:GFP and pHHO1:HHO1:GFP expressing plants (scale bars, 200 mm).

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HRS1 and HHO1 are nuclear-localized TFs involved in the control of the root elongation.

HRS1 binds to two different cis-regulatory elements. To identify cis-regulatory elements (CREs) directly targeted by HRS1 protein, we searched for conserved motifs in the promoters of the most strongly up- and down-regulated target genes. Thus, we examined the 500 bp promoter sequences upstream the transcription start site for the 120 top induced and repressed genes (based on fold regulation upon DEX treatment) from the TARGET transcriptome analysis (presented in Fig. 1f). Five strongly signicant motifs were retrieved using the MEME algorithm40. The two cis-motifs, which were over-represented in the promoters of genes upregulated by HRS1, display the consensus sequences AGANNNAAA and AAACNNAACC. By contrast, the HRS1 downregulated genes share three over represented motifs: AANNAGA, TGGGC and GAGA (Fig. 3a). Among these last three cis-motifs, the TGGGC(C/T) motif was already known as the core-binding sequence of the CRE for TCP (teosinte branched1, cycloidea, PCF) TFs41. Inspired by the consensus

sequences above, we dened ve motifs (M1M5; Supplementary Fig. 5c; Supplementary Table 1) that were tested in vitro for direct binding by HRS1 in Electophoretic Mobility Shift Assays (EMSAs). The HRS1 recombinant protein produced a shift for motifs #1, 2, 3 and 5. The shifts were completely reverted by the competition of a 200-fold molar excess of the same unlabelled probe (Supplementary Fig. 5d). No band shift was detected for motif #4, which corresponds to the TCP-binding site. This means that TCP TFs may be partners of HRS1 mediating its repressive transcriptional activity. Motifs #1, 2 and 3 in the HRS1-induced genes share the core sequence AGA and the motif #5 the core AACC (already known as part of the GLK1/2-binding site27). This is in accordance with the recent nding that a particular TF can bind to distinct cis-elements42. The specicity of HRS1 binding to the selected DNA probes was conrmed by EMSA analysis, using unlabelled probes mutated on the above described core sequences (AGA and AACC). Ten-, 25-, and 50-fold molar excess of unlabelled mutant probes could not compete with HRS1 binding to motifs #1, 3 and 5, thus conrming that HRS1 binding is maintained by AGA and AACC core sequences (Supplementary Fig. 5e).

500 bp Upstream(within 120 induced genes)

500 bp Upstream(within 120 repressed genes)

7.3e02150 Sites

1.9e01549 Sites

2.5e00847 Sites

4.4e01348 Sites

1.1e01745 Sites

P1

HRS1

P2 P3 P4 P5 P6 P7 P8-A P8-B P9A P9B P9C P10 + + + + + + + + + + + + +

Gene ID Strand Start P-value Site

AGA core

P1 AT2G28550 RAP2.7 + 445 4.16e06 TTATAGTTAC AGAGAGAGAAAA ACAACTTTAT *

P2 AT5G13910 LEP 409 6.43e06 CTAGTTGAAG GAAGAGAGGGAA TGTGTGTTGG *

P3 AT2G47810 NF-YB5 + 321 2.91e06 AAGAAAATTG AGAGAGAGAGAA ATTAGCTAGA *

P4 AT4G03270 CYCD6;1 432 4.16e06 GTTAGAAGAT GAAGAAGAAGAG AGAGGGACAG

P5 AT3G02060 DEAD 489 1.45e07 AGGGGATAAT GAAGAGAGAGAG AGAGAGAGTG *

AACC core

P6 AT2G44530 + 367 1.41e-05 AGACCTTCCG AAAATAAACCGA ATTTCATCGG

P7 AT4G35900 bZIP14 + 487 6.06e06 TGGAGTCTTG GACCCAACCCCA TC

P8-A AT2G28550 RAP2.7 + 340 1.21e05 AAAAAAAAAA AAAAAAAACCGG AAACTTACTT

P8-B AT2G28550 RAP2.7 + 84 2.24e06 GTCACCCCAA AACCCAAACCAA ACGACGTGAA *

P9-A AT5G22370 QQT1 352 1.14e07 GGAGTATCCG AACCCAAACCGA AAAGTGAATT *

P9-B AT5G22370 QQT1 415 1.34e07 TGCTATACTC GAACCGAACCGG ATAATCGAAA *

P9-C AT5G22370 QQT1 + 330 1.91e06 ATCCGAAAAC AAACCGACCCGA AATTCACTTT *

P10 AT3G14070 CAX9 440 5.35e06 ATAAACCCCA AAACCTAACCGA ACCAGAAAGG *

Figure 3 | Identication of cis-regulatory elements and binding of HRS1 to the promoters of meristem-related genes. (a) Weight matrix representation of the motifs retrieved by the MEME algorithm analysis from the 500 bp sequences upstream the transcription start sites of the top 120 direct up- and downregulated HRS1-target genes. (b) EMSA analysis on 30-bp promoter fragment from HRS1 directly regulated genes (from TARGET analysis); biotin-labelled DNA probes (20 fmol), HRS1GST protein (50 ng). (c) List of the promoter fragments sequences used for EMSA analysis in a. For each promoter the gene ID, the common name and the strand position is indicated.

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To obtain functional explanations of the root developmental phenotype described (Fig. 2ac), we tested whether HRS1 was also able to bind native CREs found in promoters of genes categorized as meristem-related genes. For that we used 30-bp promoter fragments as EMSA probes. HRS1 produced a signicant shift for 9 of 13 promoter fragments tested (Fig. 3b,c). Promoters have been selected as they are; (i) induced by HRS1 in the TARGET system (Fig. 1f); and (ii) they are classied by GO as being involved in meristematic activity. To summarize, HRS1 is shown to be able to bind the promoter sequences of RAP2-7 (related to AP2.7; AT2G28550), LEP (leafy petiole; AT5G13910), the NF-YB5 (nuclear factor Y, subunit B5; AT2G47810) TF, the helicase DEAD (AT3G02060), the DNA-binding protein QQT1 (quatre quart 1; AT5G22370) involved in embryo development, and the cation calcium exchanger CAX9 (cation calcium exchanger 9; AT3G14070).

These results show that HRS1 possesses two distinct target CREs found in HRS1-activated genes belonging to developmental processes pathways.

HRS1 is post-transcriptionally controlled by P provision. Since HRS1 is clearly strongly transcriptionally activated by the NO3

signalling pathway (Fig. 1a), and it is involved in the convergence of Pi and NO3 signals (Fig. 2), we investigated how Pi and NO3

might affect the levels of HRS1 protein. As expected for such a NO3 -induced transcript (Fig. 1), the HRS1 protein strongly accumulates in response to NO3 treatment (Fig. 4a). Interestingly, in whole roots, HRS1:GFP protein accumulation was reduced after 24 and 48 h of Pi deprivation (Fig. 4b). In the same conditions (48-h Pi starvation), HRS1 mRNA levels are not changed, even if the plant felt the Pi deprivation (as reported by IPS1, Fig. 4b). These ndings suggest a possible post-transcriptional regulation of HRS1 by Pi provision. Interestingly, this interaction of NO3 transcriptional induction of HRS1, together with its post-transcriptional regulation in response to P, could

constitute a mechanism that entangles both nutritional signals. To conrm this hypothesis, the NO3 transcriptional regulation has to be conserved independent of the preceding P treatment. To demonstrate this, we veried that the transcriptional regulation of HRS1 by NO3 is maintained in P varying conditions (Fig. 4c).

Furthermore, we observed that the accumulation of the HRS1 protein accumulation is not affected by P provision in the short-term experiments. This demonstrates that NO3 and P

signals act early (within minutes) and late (within days) at the transcriptional and post-transcriptional levels, respectively (Fig. 4). To delve further into one potential mechanism of the P effect on HRS1 activity, we analysed HRS1GFP protein half-life in P varying conditions (Fig. 5a,b). We recorded that P treat

ment shifts the HRS1 half-life from B20 to 30 min. Interestingly, this mechanism is not restricted to HRS1, since another nitrate-regulated protein is under this kind of regulation (Fig. 5c). Indeed, we have found that the nitrate transporter/sensor NRT1.1 is strongly destabilized on P conditions (as compared with

another membrane protein PIP2.1, Fig. 5c). This demonstrates that P deciency conditions may broadly affect nitrate-regulated proteins to convey a layer of the NO3 and PO43 signal interaction.

Genome-wide effect of HRS1 and HHO1 on gene expression. To better document the in planta effects from the modication of the HRS1/HHO1 TFs, plantlets (Col, hrs1;hho1, HRS1-OE and HHO1-OE) were grown in P/ NO3 conditions for 6 days in

three independent experiments. Whole roots (Fig. 6a) were harvested to measure the effect of these HRS1/HHO1 genetic modications using last generation Affymetrix chips (Arabidopsis

gene1.1ST array, Supplementary Data 2). Analysis was performed as previously described in refs 43,44 (for details see Methods). Analysis of variance (ANOVA) followed by a post hoc Tukey test yielded 1,125 HRS1/HHO1-regulated probes corresponding to 969 unambiguous genes. A clustering analysis helped to understand the dominant mode of regulation triggered by the mutation or the overexpression of HRS1 and HHO1 (Fig. 6b). Among these 969 HRS1/HHO1-regulated genes, 22 genes have been identied to be direct HRS1 targets based on the TARGET approach (11 upregulated and 11 downregulated, Supplementary Data 1). This relatively low number is however higher than one would expect by chance (Monte Carlo test P valueo0.05).

Among these HRS1 direct targets is NRT1.1, which is found in cluster #8. Since we have shown that NRT1.1 is a key regulator of

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response. (a) HRS1GFP protein accumulation is induced in response to 1 mM NO3 treatment. (b) HRS1GFP protein accumulation is affected by P provision; 48 h P starvation (reported by IPS1 sentinel regulation) does not affect HRS1 (endogenous gene), HHO1, and HRS1:GFP mRNA accumulation. (c) Rapid NO3 transcriptional activation of HRS1 and subsequent protein accumulation is maintained regardless P provision. Different NO3 levels induce: (i) HRS1GFP protein accumulation in roots of 48-h P starved plants and (ii) HRS1 transcript accumulation in plant roots grown (14 days) on P varying media. Values are meanss.e.m. (n 3).

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and P conditions. (b) Quantication of immunoblot signal. (c) P starvation accelerates the protein degradation rate of the nitrate transporter/sensor NRT1.1, but not the aquaporin PIP2.1 (used as non-nitrate-inducible control).

HRS1 primary NO3 induction (Fig. 1e), this demonstrates a potential feedback mechanism of HRS1 on its own regulator, as is often found in regulatory networks. The 969 HRS1/HHO1-regulated genes have been subjected to GO enrichment analysis by the agriGO web site39 followed by a REVIGO analysis to reduce and summarize the number of GO terms detected45. Interestingly, this analysis demonstrates that phosphorus metabolism, hormone metabolism, starch metabolism, developmental process, cellular nitrogen compound metabolism, post-embryonic development, response to red light and response to abiotic stimulus are (among others; Supplementary Fig. 6) enriched in this list. This demonstrates that (i) HRS1/HHO1 indeed affect the processes that have been predicted by the cell-based TARGET system approach (Fig. 1g; Supplementary Fig. 6), and (ii) that HRS1/HHO1 may have other functions, as the one described herein, some of which that was predicted by other studies (refs 35,46 and discussed below).

To obtain further insights into the altered gene responses triggered by altered expression of HRS1/HHO1, a clustering analysis uncovered 19 clusters of mis-regulated genes (Supplementary Data 2). The overall analysis of the 19 clusters demonstrates that even if HRS1 and HHO1 seem to play redundant roles in the control of primary root growth (Fig. 2), they may control at the same time the same set of genes (such as in cluster #9,17,7,14,10,8) or different genes (such as in clusters #4,11,12,15,13,16). AgriGO was then used to determine the molecular coherence of these HRS1/HHO1-regulated gene clusters into potential biomodules (that is, co-regulated genes having a particular function47). Interestingly, in the clusters for which HRS1 and HHO1 have a coherent effect on gene regulation (control the same set of genes), the GO terms are related to stress responsiveness and response to reactive oxygen species (ROS). Moreover, it seems that HRS1 overexpression is the one that triggers the most regulation on genes responsible for growth control (that is, cluster #11). Taken together, this could suggest that HRS1 and HHO1 play redundant functions due to their

phylogenetic proximity, but might control different set of genes depending on the genomic context. However, since the two TFs converge towards the control of ROS signalling-related genes, and that ROS have recently been shown to be involved in the control of primary root meristematic activity48 or cell size49, it is tempting to propose that such a mechanism could be downstream of HRS1/HHO1 inuence. It is however important to note that UPB1 does not belong to the 969 HRS1/HHO1-regulated genes based on whole-root transcriptome studies. These hypotheses will need further experiments to be fully validated.

One hypothesis related to the modularity of the HRS1 versus HHO1 effect is that they can affect different genes according to the possible tissue context. To document this possibility, the different HRS1/HHO1-controlled gene clusters were overlapped to a set of genes dened as markers of different root cell layers50. Interestingly, clusters #11,7,17,14,10,8,18 appear to signicantly (P valueo0.05, Monte Carlo test named GeneSect22) overlap with cell type-specic markers (third column, Fig. 6b). Thus, since the transcriptome is performed at the whole-root level, this overlap may show that some clustering properties are related to different activities of the two TFs in different cell types. Again, this will need to be fully validated by following further experimental studies.

The most striking cluster of this analysis is #8. Indeed, it gathers genes being more highly expressed in the double hrs1-1; hho1-1 mutant and repressed by HRS1 or HHO1 overexpression (Fig. 6b). Interestingly, this cluster comprises genes known to be responsive to NO3 (including NRT1.1 and NRT2.4, among others) and hormones. Indeed, response to auxin stimulus is also an over-represented GO term in this gene cluster. This observation opens perspectives concerning the role of auxin signalling in the control of root developmental mechanisms that could explain the effect of HRS1 and HHO1 in P conditions.

To consolidate the fact that HRS1 binds cis-elements of six genes that are found to be direct targets of HRS1, which are activated in the cell-based system (Figs 1 and 3), we wondered why these genes are not found in the 969 HRS1-regulated genes detected in whole roots? Since the P effect is very much localized to the root apex, and likely not affected by the whole-plant P status (according to transfer experiments Supplementary Fig. 3), we hypothesized that the regulation by P may be restricted to the most apical part of the root. To validate this hypothesis, we performed transfer experiments from P/ N to P/ N

media. The tip of the primary root (23 mm) was harvested from the WT, hrs1-1;hho1-1 double mutant and overexpressors and RNA used for qPCR experiments. This revealed that RAP2.7, NF-YB5 and the DEAD helicase are indeed regulated by HRS1 overexpression at the root apex (Fig. 6c). Several meristem-related genes (CLV1, FAS1 and TSO1), dened as direct targets by the cell-based TARGET system (Supplementary Data 1), were also measured and displayed misregulation in the hrs1;hho1 double mutant, but with no effect of the TF overexpression (Fig. 6c). This demonstrates that these genes may be indeed under the direct inuence of HRS1 in plants, and are potential candidates controlling meristematic activity together with the previously evoked pathways that include auxin and ROS signalling (refers to clusters #8 and #9,15,11, respectively, Fig. 6b). To decipher the different regulatory pathways below the action of the genes known to sensitize primary root to local P deciency, it will be important to confront whole-genome expression reprogramming induced by lpr1/2 (ref. 8), pdr2 (ref. 9) and hrs1;hho1 to dene whether these regulatory modules share common signalling pathways. Further, reverse genetic studies will also be needed to validate that the potential regulators of meristematic activity such as CLV1, FAS1, TSO1, RAP2.7 or NF-YB5 are indeed a part of the HRS1/HHO1-mediated response.

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2.5 0.0 2.5

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genes

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GO:0009791 post-embryonic development 0.0078 GO:0019825 oxygen binding 0.00044

GO:0044262 cellular carbohydrate metabolic process 0.0014

GO:0034641 cellular etc nitrogen compound metabolic process 0.017

GO:0016491 oxidoreductase activity 0.0073

GO:0009408 response to heat 2.8e05 GO:0006950 response to stress 0.00036

GO:0000302 response to reactive oxygen species 0.00088

AGL12 AGL14 AGL22 PID

LAX2 EXLB1 Phloem

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ATEXP23 pectin lyase

Cluster#

Figure 6 | Probing the effect of HRS1 and HHO1 mutations and overexpressions in P-decient root transcriptome. (a) Six-day-old plantlets were

grown on P/ NO3 conditions (b) or on P/ NO3 media and then transferred 2 days to P/ NO3 conditions (c). (b) Clustering of the

969 HRS1-regulated genes from whole roots of plantlets grown on P/ NO3 media. For each cluster, a selection of over-represented GO terms is

given, as well as remarkable genes belonging to the cluster. Clusters overlap with NO3 (ref. 31); hormonal59 responsive genes or root cell type-specic markers50 have been measured using the GeneSect algorithm22. Signicant overlaps (P valueo0.05) are reported in the third column. (c) Reverse transcription qPCR analysis on selected HRS1 direct targets on RNA extracted from 2- to 3-mm sections of the root apex in a transfer experiment.

Values are meanss.e.m. (n 3). Residual HHO1 expression in the mutant is explained in Supplementary Fig. 2.

Finally, we believe that this transcriptomic analysis provides an important proxy towards understanding the other roles of HRS1 in plants. Indeed, it has been shown to be controlling germination51, or to confer drought or salt stress tolerance52. Again the general functions regulated by HRS1 such as ROS signalling, as well the important overlap with ABA responsive genes are important keys to understand the underlying mechanisms behind the previously reported phenotypes.

DiscussionAs we take together the above observations, we propose the model described in Fig. 7. HRS1 is under a dual regulation: (i) a NO3 -induced (NRT1.1/CHL1 NLP7 dependent) transcriptional control, and (ii) a Pi-regulated post-transcriptional control. In response to these two signals, HRS1 controls primary root growth through possibly: (i) its action on two independent CREs contained in promoters of activated genes; and (ii) its effect on

pathways such as ROS or Auxin signalling. These two possibilities are not exclusive. Our observations can, to some extent, be related to the recent work of Kellermeier et al.53, which makes a clear demonstration that nutrient signalling interaction is rather a general rule than an exception.

Our work provides a potential mechanism for understanding how the two key plant mineral nutrients (PO43 and NO3 )

interact to control root growth, an issue of global importance, since nitrogen and phosphorus are the two most widely used fertilizers worldwide that maintain plant growth and production. For instance, a recent work by Delgado-Baquerizo et al. showed that at the ecological level, drying lands might impact markedly nitrogen and phosphorus ratios available for plants, thus jeopardizing food production worldwide54. The molecular events, mediated by HRS1 and HHO1 in plants, that integrate two mineral-related signalling pathways are part of the fundamental knowledge needed to tackle such key environmental challenge in a global warming context.

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Figure 7 | Proposed model: NO3 and P limitation signals are integrated by the control of HRS1 at the transcriptional and post-transcriptional level, respectively. On NO3 treatment, the NRT1.1 and NLP7 regulatory module induces rapid HRS1 transcript and protein accumulation. The prolonged P deciency condition negatively affects the accumulation of HRS1 and modies its regulatory activity. Furthermore, the P limitation signal seems to control the NO3 sensor (NRT1.1) protein half-life. This delineates a working model to understand several points of NO3 and

PO43 signal interactions via the HRS1 pathway.

Methods

Plant material. All A. thaliana plants were in the Columbia background. Mutant hrs1-1 (SALK_067074), hho1-1 (SAIL_28_D03) and mutant chl1-12 (Salk_034596) were obtained from ABRC seeds stock center. The hrs1-1;hho1-1 double mutant has been obtained by crossing. Despite the presence of a highly accumulated chimeric RNA in the hho1-1 line (characterized in Supplementary Fig. 2c), the production of a full-length gene product is knocked out (Supplementary Fig. 2b). The promoter gene GFP lines for tissue localization were obtained by cloning HRS1 and HHO1 from genomic sequences, bringing respectively the 3- and 2.5-kb upstream promoter region and the gene, into pMDC107 Gateway-compatible vector. Overexpressor lines were obtained by cloning HRS1- and HHO1-coding sequences into pMDC32 Gateway-compatible vector55, using primers listed in Supplementary Data 1. The constructs were transferred to Agrobacterium tumefaciens strain GV3101 and used for the Arabidopsis transformation by the oral dip method56. Transgenic plants were selected by antibiotic resistance and T3 homozygote descendants were used for analysis.

Growth conditions and treatments. For NO3 treatment experiment, plants were grown in sterile hydroponic conditions as described in ref. 22. Hydroponic media consisted of MS basal salt medium containing no nitrate and supplemented with 3 mM sucrose, 0.5 mM ammonium succinate, MES buffered at pH 5.7 (0.5 g l 1).

Plants were grown for 15 days in day/night cycles (16/8 h; 65 mmol photonsm 2 s 1) at 22 C. Plants were transferred to an equivalent fresh nitrogen-free medium for 24 h and then treated with nitrogen as 1 mM KNO3 or 1 mM KCl, as mock treatment. Roots were sampled at different time points after the treatment and immediately frozen in liquid nitrogen. For P starvation experiments, Col-0 and hrs1-1;hho1-1 seeds were sown on the surface of solid media consisting of MS basal salt medium nitrogen and phosphorous-free supplemented with KNO3 at different concentrations (0.05, 0.5, 1 and 2.5 mM), 0.5 mM KH2PO4 for P-sufcient condition, 3 mM sucrose, MES (0.5 g l 1) and 0.8% (w/v) agarose. Different volumes of 1 mM KCl solution were added to the media to keep the K concentration constant among different conditions. Plants were grown vertically for 9 days in day/night cycles (16/8 h; 90 mmol photons m 2 s 1) at 22 C.

For P starvation experiments in liquid media, plants were grown for 15 days in sterile hydroponics conditions, in MS media containing 5 mM KNO3 and 1 mM

KH2PO4. Plants were transferred towards an equivalent KH2PO4-free fresh medium ( P) or 1 mM KH2PO4 media ( P) for 48 h. For half-life experiment,

CHX was used at 100 mM. All growth and gene expression experiments are supported by at least three independent experiments.

Root growth measurements. Beginning 2 days after seed sowing, two Petri dishes per condition (that is, at least 30 plants) were scanned every day at 400 d.p.i. Root length was measured using Optimas 6.1 software and statistical differences between genotypes were calculated using Students t-test.

Real-time qPCR analysis. Total RNA was isolated from Arabidopsis roots and shoots using TriReagent (Molecular Research center Inc.) and digested with DNAseI (Sigma-Aldrich, St Louis, USA). Total RNAs were then reverse transcribed to one-strand complementary DNA using Thermo script RT (Invitrogen) according to the manufacturers protocol. Gene expression was determinedby reverse transcription qPCR (LightCycler 480; Roche Diagnostics, Basel, Switzerland) using gene-specic primers (listed in Supplementary Data 1) and LightCycler 480 SYBR Green I Master mix (Roche, IN, USA). Expression levels of tested genes were normalized to expression levels of the actin and clathrin genes.

TF perturbation assays in the TARGET system. The TARGET procedure has been performed as previously described in Bargmann et al.36 Protoplasts were treated with 35 mM CHX and 10 mM DEX. The red uorescent protein was used as marker selection for uorescent-activated cell sorting of successfully transformed protoplasts. One milimolar NO3 was maintained during the whole procedure.

RNA was extracted and amplied for hybridization with ATH1 Affymetrix chips. Data were analysed with R. Mas5 normalized data were extracted and analysed by a t-test procedure for DEX response (P valueo0.05, fold regulation42 ,

corresponding to a false discovery rateo0.1).

Microscopy. Col-0, pHRS1:HRS1:GFP and pHHO1:HHO1:GFP seedlings were grown on half-strength MS media for 8 days. The same liquid media was used for mounting plantlets during the image capture. Fluorescence imaging was performed using the Olympus BX61 microscope. Samples were excited at 470 nm and emission was collected between 500 and 535 nm. Confocal images were performed with a Zeiss LSM510 Meta laser scanning microscope. Root cell walls were stained with propidium iodine 10 mg ml 1 (Sigma) and nucleus with 4,6-diamidino-2-phenylindole 1 mg ml 1 (Sigma). Three-dimensional root reconstructions were obtained from confocal Z series using the Imaris software.

Semantic gene enrichment analysis. To identify the most represented gene functions for each gene list, the gene descriptions of targets were retrieved from TAIR web site (http://www.arabidopsis.org/index.jsp

Web End =http://www.arabidopsis.org/index.jsp) and the list was analysed using a software developed in R (http://www.r-project.org/

Web End =http://www.r-project.org/). Briey, given the list of 551 HRS1 up-/downregulated genes, the software counts the occurrence of each term of the description and compares it with the occurrence of the same term in 1,000 random lists of the same size. Results are presented as a cloud of words, whose colour and size are correlated to their occurrence.

Phylogenetic analysis. The phylogeny reconstruction was inferred by using the maximum likelihood method. The 40 sequences coding for G2-like proteins were retrieved from the AGRIS (Arabidopsis Gene Regulatory Information Server; http://arabidopsis.med.ohio-state.edu/

Web End =http://arabidopsis.med.ohio-state.edu/ ) database. The bootstrap values were obtained based on 500 replicates. Evolutionary analysis was conducted in MEGA5 software57.

Expression and purication of recombinant GST-HRS1 protein. HRS1 coding sequence CDS was rst cloned in pDONR207, using the primers listed in Supplementary Data 1 and then transferred to pDEST15 vector (Invitrogen) by LR reaction following the manufacturers instructions. The GST-HRS1 fusion protein was expressed in Escherichia coli Rosetta 2(DE3)pLysS (Novagen, Darmstadt, Germany) cells after induction with 1 mM isopropyl-b-D-thiogalactoside for 16 h at 22 C. Bacteria were harvested, suspended in PBS buffer containing lysozyme from chicken egg white (Sigma) and complete protease inhibitor cocktail (Roche) and sonicated. The protein extract was puried on glutathione sepharose beads (GE Healthcare, Freiburg, Germany), eluted with 10 mM reduced glutathione (Sigma), 50 mM Tris buffer and dialysed overnight in 150 mM NaCl, 50 mM HEPES, pH 7.4 buffer.

EMSA. Puried GST-HRS1 recombinant protein was used to determine DNA binding by EMSA. Single-stranded DNA oligonucleotides (listed in Supplementary Data 1) were biotin labelled using the Biotin 30 End DNA Labelling Kit (Thermo

Scientic) and complementary pairs were annealed to make double-stranded DNA probes. The binding of recombinant protein (50 ng) to the biotin-labelled probes (20 fmol) was carried out in a reaction mixture containing 10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5, 2.5% glycerol, 5 mM MgCl2, 1 mg poly (deoxyinosinic

deoxycytidylic) and 0.05% NP-40. After incubation at 22 C for 30 min, the proteinprobe mixture was separated in a 4% polyacrylamide native gel and transferred to a Biodyne B Nylon membrane by capillary action in 20 SSC buffer

overnight (Thermo Scientic). After ultraviolet crosslinking (254 nm) for 90 s at 120 mJ cm 2, the migration of biotin-labelled probes was detected using strep

tavidin horseradish peroxidase conjugates in the Chemioluminescent Nucleic Acid

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Detection Module (Thermo Scientic) and exposed to X-ray lm. As a negative control, we tested that the GST tag has no afnity for HRS1-related CREs (Supplementary Fig. 5).

Protein extraction and immunoblot analysis. Roots were sampled and frozen in liquid nitrogen. Total proteins were obtained using the Plant Total Protein Extraction Kit (Sigma) and quantied with the Pierce 660 nm Protein Assay using a BSA standard curve. Membrane proteins were obtained by disrupting roots ina buffer containing a plant anti-protease cocktail (Sigma-Aldrich) and anti-phosphatases (30 mM glycerophosphate, 5 mM molybdate and 10 mM NaF). The whole-membrane fraction was then isolated by centrifugation (100,000 g, 4 h) on a 55% sucrose cushion. Immunoblot analysis was performed on 4050 mg of proteins using anti-GFPHRP 1:2,500 (Miltenyi Biotec, 130-091-833), anti-NRT1.1 1:5,000 (AS12 2611, Agrisera) and anti-PIP2.1 1:5,000 (ref. 58). Coomassie Brilliant Blue staining of blots was used to control the protein levels after electrotransfer. Uncropped versions of the blots are provided in Supplementary Figs 7 and 8. Band intensity quantication was performed using a chemioluminiescent image analyser LAS3000 (Fujilm) and ImageGauge (Fujilm) software.

Transcriptome analysis. The transcriptome analysis was performed using the GeneChip Whole Transcript (WT) Expression Array following the manufacturers protocol. Total RNA was isolated from 6-day-old roots grown on media containing2.5 mM KNO3 and no PO43 (three independent experiments). RNA integrity numbers were checked by microuidic analysis in an Agilent 2100 Bioanalyzer.

Complementary DNAs were prepared from 150 ng of total RNA following the WT Plus Reagent Kit protocol, hybridized on Affymetrix Array Strip and processed on the GeneAtlas System. Data set analysis was performed with R. A one-way ANOVA model was applied (four levels: Col, hrs1-1;hho1-1, HRS1-OE and HHO1-OE) followed by a post hoc Tukey test (R functions aov() and TukeyHSD()). Any probe having a signicant P valueo0.05 for the ANOVA or the Tukey test was kept for further analysis. Clustering analysis was performed with MeV software (distance, Pearson correlation).

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Acknowledgements

We thank Benot Lacombe and Frederic Gaymard for the comments and discussions; Elodie Jublanc from the Montpellier Rio Imaging (MRI) core facility for the confocal assistance and three-dimensional imaging reconstruction; Tony Sierra for the technical assistance during Gene Atlas analysis; Veronique Santoni for the PIP2.1 antibody; and Franck Lecocq, Rogatien Picaud, Guy Ruiz, Thierry Dessup, and Hugues Baudot for the technical support and plant care. This work was supported by the French Agence Nationale de la Recherche (NitroNet: ANR 11 PDOC 020 01) and Centre National de la Recherche Scientique (PEPS Bio math Info 20122013: SuperRegNet) to G.K. Work on the transcriptional networks was supported NIH R01-GM032877 to G.C. and NIH NRSA-GM095273 to A.M.-C. Results on root architecture responses are supported by NSF MCB-0929338 to G.C. Bioinformatics analysis was supported by the VirtualPlant platform (http://www.virtualplant.org

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Author contributions

G.K. designed the project. A.M., A.M.-C., E.R., W.S., R.W., S.R. and G.K. performed the experiments and analysed the data. A.M., A.G., N.M.C., S.R., G.M.C. and G.K. contributed to the study design during the project course. A.M. and G.K. wrote the paper.

Additional information

Accession codes: Microarray data associated with this study has been deposited in NCBI GEO database under the accession code GSE64352.

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How to cite this article: Medici, A. et al. AtNIGT1/HRS1 integrates nitrate and phosphate signals at the Arabidopsis root tip. Nat. Commun. 6:6274doi: 10.1038/ncomms7274 (2015).

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