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# Nitrate acts at the Arabidopsis thaliana shoot apical meristem to regulate flowering time

Justyna Jadwiga Olas, Judith van Dingenen, Christin Abel, Magdalena Anna

Dzialo, Regina Feil, Anne Krapp, Armin Schlereth, Vanessa Wahl

► To cite this version:

Justyna Jadwiga Olas, Judith van Dingenen, Christin Abel, Magdalena Anna Dzialo, Regina Feil, et al. Nitrate acts at the Arabidopsis thaliana shoot apical meristem to regulate flowering time. New Phytologist, 2019, 223 (2), pp.814-827. 10.1111/nph.15812. hal-02625635

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# Nitrate acts at the *Arabidopsis thaliana* shoot apical meristem to regulate flowering time

Justyna Jadwiga Olas¹ ID, Judith Van Dingenen¹ ID, Christin Abel¹ ID, Magdalena Anna Działo¹ ID, Regina Feil¹ ID, Anne Krapp² ID, Armin Schlereth¹ ID and Vanessa Wahl¹ ID

¹Department of Metabolic Networks, Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476, Potsdam, Germany; ²Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles, France

Authors for correspondence:
Vanessa Wahl
Tel: +49 331 456 8116
Email: vanessa.wahl@mpimp-golm.mpg.de

Armin Schlereth
Tel: +49 331 456 8114
Email: schlereth@mpimp-golm.mgp.de

Received: 9 November 2018
Accepted: 15 March 2019

New Phytologist (2019) 223: 814–827

doi: 10.1111/nph.15812

**Key words:** flowering time, NIN-LIKE PROTEINS (NLPs), nitrate, nitrate-responsive elements (NREs), shoot apical meristem (SAM), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1), trehalose 6-phosphate (T6P).

## Introduction

Plants take up nutrients from soil throughout their life cycle, providing the potential to grow in tight coordination with the developmental demand (Krouk *et al.*, 2011). This is crucial for the timely transition between developmental phases such as the switch from the vegetative to the reproductive phase (Lin & Tsay, 2017). Nitrogen (N) is an essential macronutrient for plant growth, and high yields of almost all cultivated crops require application of large amounts of fertilizer. Hence, N plays an important role in agriculture to improve yield and increase agronomical productivity. However, although fertilizers have helped to increase yield, they also have negative effects, causing environmental and human health problems as well as decreased biodiversity (Smil, 1999; Good & Beatty, 2011; Shibata *et al.*, 2015). A surplus of synthetic fertilizers and manure causes emission of nitrous oxide into the atmosphere and leaching of nitrate into soil patches. Many natural water basins and aquifers around the world are currently steering towards severe nitrate pollution through long and continued application of organic and synthetic fertilizers. The maximum contaminant level for public water supply of 50 mg nitrate $l^{-1}$ determined

by the World Health Organization (or 10 mg $l^{-1}$ nitrate-N, stipulated by the US Environmental Protection Agency) is being well exceeded (Ward *et al.*, 2018). Reducing the application of fertilizers is a general aim of modern agriculture.

Nitrate is the major source of N for plants and, once taken up by the roots, is distributed within plants by a large number of nitrate transporters. Besides being a nutrient, nitrate itself acts as a signal regulating directly the expression of hundreds of genes (reviewed in Noguero & Lacombe, 2016). These genes encode proteins required for nitrate transport and assimilation, and the reprogramming of carbon (C) and N metabolism, as well as transcription factors and regulatory proteins, triggering a cascade of changes that support increased growth. An early event in nitrate responses involves the accumulation of the transcription factor NIN-LIKE PROTEIN 7 (NLP7) and its close homolog, NLP6, in the nucleus (Marchive *et al.*, 2013; Guan *et al.*, 2017), where they bind to nitrate-responsive elements (NREs) in promoter regions of target genes (Konishi & Yanagisawa, 2013) and regulate their expression.

Yield depends not only on vegetative growth, but also on the optimal timing of reproductive growth. The latter starts with the

## Summary

* Optimal timing of flowering, a major determinant for crop productivity, is controlled by environmental and endogenous cues. Nutrients are known to modify flowering time; however, our understanding of how nutrients interact with the known pathways, especially at the shoot apical meristem (SAM), is still incomplete. Given the negative side-effects of nitrogen fertilization, it is essential to understand its mode of action for sustainable crop production.

* We investigated how a moderate restriction by nitrate is integrated into the flowering network at the SAM, to which plants can adapt without stress symptoms.

* This condition delays flowering by decreasing expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) at the SAM. Measurements of nitrate and the responses of nitrate-responsive genes suggest that nitrate functions as a signal at the SAM. The transcription factors NIN-LIKE PROTEIN 7 (NLP7) and NLP6, which act as master regulators of nitrate signaling by binding to nitrate-responsive elements (NREs), are expressed at the SAM and flowering is delayed in single and double mutants. Two upstream regulators of SOC1 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and SPL5) contain functional NREs in their promoters.

* Our results point at a tissue-specific, nitrate-mediated flowering time control in *Arabidopsis thaliana*.
transition of the shoot apical meristem (SAM) from a vegetative
into an inflorescence meristem. This developmental switch is reg-
ulated by a complex hierarchical signaling network that integrates
many environmental and endogenous stimuli (Blumel et al.,
2015). Within this network, core pathways have been described
that orchestrate responses to day length and light quality (pho-
toperiod pathway), fluctuations in temperature (ambient temper-
ature pathway), exposure to longer periods of cold
(vernalization), gibberellic acid signaling (GA pathway), endoge-
nous regulators independent of light and GA (autonomous path-
way), and the plant's age (age pathway). Although these signaling
pathways have been extensively studied, knowledge of how
metabolic signals regulate flowering lags behind. A notable excep-
tion is the dependence of the induction of flowering on a plant's
energy status. The trehalose 6-phosphate (T6P) pathway has been
shown to convey information about the sucrose status to the
flowering network at two signal perception sites. In the leaves, it
is necessary and sufficient to induce FLOWERING LOCUS T
(FT), also known as the florigen and a member of the photope-
riod pathway, at the end of long days. At the SAM, the T6P path-
way interacts with the age pathway both via miR156 and
independently of it (Wahl et al., 2013).

The nitrate supply is known to modify several developmen-
tal processes, including flowering time (Rideout et al., 1992;
Corbesier et al., 2002; Castro Marin et al., 2011; Liu et al.,
2013). The first evidence suggesting that nitrate might be
involved in the regulation of flowering time in Arabidopsis
thaliana was obtained from genetic studies showing that nitrate
assimilation and signaling/uptake mutants are late-flowering
(Tocquin et al., 2003; Seligman et al., 2008). However, inter-
pretation of these observations is challenging as these mutants
display severe global metabolic changes, including sugar con-
tent, all of which can affect flowering time (Scheible et al.,
1997; Klein et al., 2000). Indeed, studies conducted with A.
thaliana under various conditions reveal that nitrate has con-
trasting effects on flowering time depending on the growth sys-
tem (agar-, vermiculite-, soil-based) and the source of nitrate
used (KNO3, NH4NO3, mixed, supplemented with glutamine
(Castro Marin et al., 2011; Kant et al., 2011; Liu et al., 2013;
Yuan et al., 2016; Lin & Tsay, 2017; Gras et al., 2018)).
Hence, depending on the study, conflicting results were
obtained regarding which flowering genes show differential
expression in different N regimes, or which flowering pathway
mutants alter the response to N, whereas various flowering
genes have been reported as correlating with the growth system
used but not necessarily responding to nitrate directly (Lin &
Tsay, 2017). In a review on the current status of research
regarding N-dependent control of flowering, Lin & Tsay
(2017) compiled data published by early 2017 and proposed a
U-shaped response of flowering to nitrate, with an optimal
nitrate concentration range for flowering, which is delayed by
higher and suboptimal nitrate concentrations (Lin & Tsay,
2017). In addition, flowering is promoted by nitrate starvation
(Lin & Tsay, 2017), with the latter being an extreme condi-
tion revealing a plant's escape strategy when exposed to stress.
Acute stress can either induce or delay flowering (Kazan &

Lyons, 2016; Takeno, 2016), defining an emergency exit to
secure next generations, should the environmental conditions
be too harsh to allow adaptation. A recent review (Takeno,
2016) suggests a separate pathway for stress-induced flowering,
which responds to diverse unsuitable conditions (e.g. poor
nutrition, UV light exposure or drought). All these flower-
inducing 'stresses' have in common the fact that their corre-
sponding signals, as different as they might be, converge with
the flowering network in leaves at the level of *FT*. Taking this
into account, Castro Marin *et al.* (2011) supplemented their
agar growth system with 4 mM glutamine, in order to be able
to vary nitrate but avoid acute N starvation.

Optimal nitrate concentrations differ between growth sys-
tems and for species. Furthermore, the concentrations are likely
to vary between various tissue types. This might explain why
various research groups have proposed different perception sites
for nitrate in the flowering time network (Castro Marin et al.,
2011; Liu et al., 2013; Gras et al., 2018). Additionally, most
previous studies were restricted to analyses on whole rosettes or
seedlings instead of tissue-targeted approaches (e.g. in the
SAM) and reflect a great variability in the light and tempera-
ture regimes used to grow the plants. Previous research pro-
vided evidence that nitrate, once taken up by plants, might
interfere with the known flowering network in leaves to mod-
ify the flowering time, as a set of flowering genes expressed in
leaves (e.g. *FT*, *SMZ*, *SNZ*) was affected by the N status (Cas-
tro Marin et al., 2011; Lin & Tsay, 2017; Gras et al., 2018).
However, no evidence has been provided on whether nitrate
enters and operates at the SAM.

Tschoep et al. (2009) established an N-limited soil system, which involves growth of plants in an ‘optimal’, full-nutrition (ON) or ‘low’ nitrate (LN) soil. The concentration of the latter was chosen in such a way that plants were able to adapt to the conditions and maintained a reduced but constant growth over several weeks. This was reflected by the metabolic phenotype, with similar amounts of protein to ON-grown plants (hereafter ON plants). Analyses of other nutrients, which might potentially affect growth and flowering time (e.g. phosphate or sulfate), demonstrated that their content was unaltered. LN-grown plants (hereafter LN plants) did not show any visible stress symptoms, such as anthocyanin accumulation, and ON-grown plants flowered at a similar time to wild-type (Col-0) plants on standard soil. Hence, this growth system is an ideal one for studying the effects that N availability might have on flowering time in *A. thaliana*.

Here, we report on a study that made use of the growth system established by Tschoep et al. (2009). We found that nitrate is present in the SAM and that nitrate-regulated genes involved in N assimilation respond in the SAM to N availability. Our data indicate that nitrate acts on the expression of components of the age pathway (i.e. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3) and SPL5) via NRE motifs in their promoters to induce flowering, and that this process involves the NLP transcription factors NLP6 and NLP7 in the SAM, and leads to differential expression of the flowering integrator gene *SOC1*.
Materials and Methods

Plant material

The plants used in this study were *A. thaliana* Columbia (Col-0, CS76778) wild-type and mutant and transgenic lines such as *co-10*, *fd-3*, *soc1-6*, *ft-10*, *tsf-1*, *ft-10/tsf-1*, *35S::amiRTPS1 35S::MIR156*, *nlp7-1* and *nlp7-1 nlp6-2* (Michaels & Amasino, 2001; Rosso *et al.*, 2003; Abe *et al.*, 2005; Michaels *et al.*, 2005; Schwab *et al.*, 2005; Yoo *et al.*, 2005; Laubinger *et al.*, 2006; Lee *et al.*, 2007; Jang *et al.*, 2008, 2009; J. W. Wang *et al.*, 2009; Wahl *et al.*, 2013). Genotypes were confirmed (Supporting Information Table S1). Cassettes containing synthetic promoters with four copies of the respective NRE (gene synthesis; Eurofins, Ebersberg, Germany) were designed as previously described (Konishi & Yanagisawa, 2010).

Growth conditions

Plants were grown in growth chambers (Percival Scientific Inc., Perry, IA, USA) at 22°C under long-day (LD; 16 h : 8 h, light : dark) or short-day (SD; 8 h : 16 h, light : dark) conditions with 160 µmol m⁻² s⁻¹ light intensity (Philips F17T8/TL841/Alto). A shift from SD to LD conditions was used to induce flowering as previously described (Schmid *et al.*, 2003).

A modified soil-based N-limited growth system (ON or LN) was used to grow plants (Tschoep *et al.*, 2009). Soil mixtures were stored for at least 2 wk at 10°C before use. The ON and LN soils contained *c*. 31.5 and 1.25 mg inorganic N per 6 cm pot (100 ml or 36 g soil per pot), respectively.

Phenotypic analyses

Flowering time was defined as days to bolting (DTB) and the total number of leaves (TLN) (Table S2). At least 11 genetically identical plants were used to determine flowering time. Student's *t*-test was used to test the significance of differences. The leaf initiation rate was calculated by dividing TLN by DTB or recording the number of visible leaves (> 2 mm) (Wang *et al.*, 2008). Juvenile leaves were defined as leaves without abaxial trichomes (Telfer *et al.*, 1997).

qRT-PCR

The total RNA was extracted using a modified phenol : chloro-
form : isoamyl alcohol (25 : 24 : 1) method as previously
described (Wan & Wilkins, 1994). cDNA preparation and quan-
titative reverse transcription polymerase chain reaction (qRT-
PCR) measurements were performed as previously described
(Wahl et al., 2013). qRT-PCR analyses were performed on two
to four biological replicates with three or four technical replicates
using the Power SYBR® Green-PCR Master Mix (Applied
Biosystems/Life Technologies, Waltham, MA, USA). Relative
quantification of gene expression was performed using a compar-
ative cycle threshold (CT) method (Livak & Schmittgen, 2001)
with a reference gene index (RGI) and presented as previously
described (Wahl et al., 2013). Primer sequences are listed in
Table S1.

GUS detection, RNA *in situ* hybridization and histological staining

For GUS reporter gene detection, seedlings were harvested in 90% ice-cold acetone, and washed with and incubated in staining buffer (10% Triton X-100, 0.5 M NaPO₄, 100 mM K-ferrocyanide, 100 mM ferricyanide, 100 mM X-Gluc) overnight at 37°C. Seedlings were dehydrated in an ethanol series, fixed with FAA (formaldehyde, ethanol, acetic acid), washed in 70% ethanol before being embedded in wax. For RNA *in situ* hybridization, apices of plants were collected at the end of the day, fixed and embedded using a Leica system (ASP300S, EG1160; Wetzlar, Germany). Sections of 8 µm thickness were prepared using a rotary microtome (RM2265; Leica). Probes were generated from cDNAs as previously described (Wahl *et al.*, 2013). For *LFY*, *SOC1*, *SPL3*, *SPL4* and *SPL5*, full open reading frames containing plasmids were provided by Weigel's and Schmid's laboratories (Weigel *et al.*, 1992; R. Wang *et al.*, 2009). RNA *in situ* hybridizations and toluidine blue staining was carried out as previously described (Wahl *et al.*, 2013).

Search for NREs in core flowering time genes

Nitrate-responsive elements (tGACcCTTNₓAAGagtcc) (Konishi & Yanagisawa, 2010) were searched in the sequences of upstream intergenic regions of known flowering genes with a maximum distance of 3000 bp to the ATG codon. A list of putative NREs is presented in Table S3 and all genes included in the analysis are provided in Table S4.

Metabolite and nitrate reductase activity measurement

For metabolite measurements, plants were harvested at end of the
day and metabolites were analyzed as previously described
(Scheible *et al.*, 1997; Nunes-Nesi *et al.*, 2007). Nitrate was mea-
sured in a coupled enzymatic assay (Cross *et al.*, 2006). An
adapted protocol was used for SAM samples, from which nitrate
was extracted with 70% ethanol once (Stitt *et al.*, 1989). Total
protein was extracted as previously described (Hendriks *et al.*,
2003). Enzyme extraction was performed with some modifica-
tions of the buffer ingredients (without triton and BSA) (Gibon
*et al.*, 2004). The extract was also used for the measurement of
total proteins, which were assayed with the Bio-Rad Bradford
reagent according to the manufacturer's instruction (Bio-Rad).
For T6P measurement, tissue samples were extracted and mea-
sured as previously described (Lunn *et al.*, 2006).

Results

Flowering is delayed when nitrate supply is suboptimal

We used a N-limited soil system (Tschoep et al., 2009) (Fig. 1a).
ON contains nitrate at a comparable concentration to standard
full-nutrition soil (31.5 mg N per pot) and supports the normal
life cycle of wild-type plants (Tschoep et al., 2009). Flowering
times for ON-grown wild-type plants (Table S2) are comparable
with data from studies using standard full-nutrition soil (± 21 d after germination (DAG) in LD and 61 DAG in SD conditions (Hartmann *et al.*, 2000; Lim *et al.*, 2004; Kim *et al.*, 2007; Wahl *et al.*, 2013), demonstrating that the selected nitrate concentration in ON is optimal for plant growth.

Plants growing in LN (1.25 mg N per pot) did not display any stress symptoms (e.g. chlorotic leaves or decreased protein and amino acid contents (Figs 1b,c, S1a)), which usually mask direct N-dependent responses (Tschoep *et al.*, 2009). Expression analyses of a stress-responsive gene by qRT-PCR did not result in any significant changes between ON and LN plants (Rowan *et al.*, 2009) (Fig. S1b). Compared with ON plants, rosettes of LN plants have significantly lower nitrate concentrations (Fig. 1d),

are smaller (Fig. 1e) and have a delay in flowering time, with a more profound effect under SD (± 16 d; Fig. 2a; Table S2) than under LD conditions (± 6 d; Fig. 2a; Table S2). When we shifted plants from SD to LD conditions at 30 DAG, a time at which plants reached competence to fully initiate flowering (Schmid *et al.*, 2003; Torti *et al.*, 2012), LN plants delayed the floral transition by only 2 d as demonstrated by RNA *in situ* hybridization using the floral marker *LEAFY* as a probe (Fig. 1f) and morphologically using stained sections (Fig. S1c). This indicates that LN conditions delay flowering and this effect can be partially overridden by the photoperiod pathway.

The timing of the floral transition at the SAM, as visualized by the increased size of the meristem at transition and the

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Physiological and morphological analyses of *Arabidopsis thaliana* wild-type (Col-0) plants grown in two nitrogen (N) regimes. (a) Simplified schematic model of the previously established, almost natural, soil-based growth system (Tschoep *et al.*, 2009), in which plants are grown in the white peat-based soil substrate containing a low (1.25 mg per pot, LN) or optimal (31.5 mg per pot, ON) N content. (b–d) Metabolites measured in rosettes of plants grown continuously in short-day conditions (SD; 8 h light : 16 h dark) or in SD to long-day (LD; 16 h light : 8 h dark) shift experiments for which plants are grown in SD for 30 d and shifted to LD for 3, 5 and 7 d to induce photoperiod-dependent flowering. Samples were harvested at the end of the day. Proteins (b) and total amino acids (c) displayed only subtle changes in LN plants. Nitrate concentrations (d) were reduced in LN plants in both sets of experiments. (e) LN plants flowered later than ON plants in all experiments, as demonstrated by flowering time analyses (here for LD grown plants, 25 d after germination (DAG)). (f) Subjecting plants to a SD-to-LD shift readily induced *LEAFY* (*LFY*) in ON plants and delayed its expression in LN plants, as demonstrated by RNA *in situ* hybridization. (g) Toluidine blue-stained longitudinal sections through apices of plants grown in SD conditions demonstrated that floral transition is largely delayed in LN plants compared with ON plants. DAS, days after shift to LD. Error bars indicate SD; statistical significance was calculated using Student's *t*-test: *, *P* < 0.05; **, *P* < 0.01; ***, *P* < 0.001. Bars: (e) 1 cm; (f, g) 100 µm.</figcaption></figure>
appearance of flower primordia, was comparable to the bolting
times we determined for plants grown on either ON or LN with,
on average, 16 d difference between the two conditions under SD
conditions (Fig. 1g). Similar to previous reports (Tschoep *et al.*,
2009; Castro Marin *et al.*, 2011; Lin & Tsay, 2017) we found
that nitrate supply to plants had an effect on the plastochron
length and hence on the production of new leaves, masking the
delay when flowering is determined based on TLN instead of
DTB (Table S2; Fig. S2). We therefore further determined flow-
ering time based on DTB.

We investigated whether growth in LN also affects the juve-
nile-to-adult phase transition as previously suggested (Vidal *et al.*,
2014). This suggestion was based on induction of the expression

of some of the miR156 precursors by acute nitrate starvation in
whole seedlings (Pant *et al.*, 2009; Krapp *et al.*, 2011; Liang *et al.*,
2012). A delay in the vegetative phase change might explain, at
least partially, the late-flowering phenotype of LN plants. We
monitored the vegetative phase change morphologically. The
number of juvenile leaves was not altered (Fig. S3a-c), demon-
strating that the length of the juvenile phase does not differ
between ON and LN plants. However, the levels of two miR156
precursors, *MIR156A* and *MIR156C*, were increased in LN-
grown rosette samples (Fig. S3d). We therefore also analyzed
abundance of *SPL* transcripts, which are the targets of miR156
(Rhoades *et al.*, 2002). SPLs belong to a large family of transcrip-
tion factors, several of which play a role in age-dependent

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Nitrate regulates flowering time at the shoot apical meristem (SAM) of *Arabidopsis thaliana*. (a) Whereas wild-type plants are significantly later-flowering in long-day (LD) and short-day (SD) conditions when grown in N-limited soil (LN), soc1-6 plants flower at the same time, indicating that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) is required for the regulation of nitrate-dependent flowering. (b, c) LN growth causes reduced SOC1 expression in the SAM of 30-d-old SD-grown plants shifted to LD for 3 and 7 d to induce flowering and harvested at the end of the day, as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) (b) and by RNA *in situ* hybridization (c) using a specific SOC1 probe on longitudinal sections through apices (compare arrows in optimal N soil (ON) vs LN at 3 d after shift (DAS)). (d) The strong expression of NITRATE REDUCTASE 2 (NIA2) in the center of the SAM, demonstrated by RNA *in situ* hybridization using a specific NIA2 probe on longitudinal sections through apices of ON-grown plants (closed arrow heads in ON), indicates that nitrate assimilation can take place in the SAM. The NIA2 expression domain is smaller in LN plants (d, open arrow heads in LN). (e) Lower transcript abundance of NIA1 and NIA2 was confirmed by qRT-PCR. (f, g) Nitrate reductase activity (NR) (f) and nitrate (g) measured at the SAM were significantly reduced in LN plants. Both NR and nitrate were calculated on the basis of protein measured in the same extracts, for which no difference was found between the treatments. Error bars denote SD; the statistical significance between ON and LN was calculated using Student's *t*-test: *, *P* < 0.5; **, *P* < 0.01; ***, *P* < 0.001. Bar, 100 µm.</figcaption></figure>
developmental transitions such as the vegetative phase change and the floral induction as part of the age pathway (Xu *et al.*, 2016). We did not detect significant changes between ON and LN plants in rosettes, except for increased levels of *SPL4* in LN plants (Fig. S3e). Increased expression of *SPL4* in the rosettes of LN plants is inconsistent, with a delayed vegetative phase change; whereas miR156 levels decline when plants age, *SPL* transcript abundances increase (Wang, 2014). Together, these results show that limiting N in a nonstressful way delays flowering time but not the vegetative phase change.

## SOC1 is required for the regulation of N-dependent flowering

To determine the potential contribution of known flowering pathways to nitrate-dependent flowering, we performed flowering time analyses of mutants in components of different pathways of the flowering network and wild-type plants grown in the two N regimes (Table S2). Interestingly, within our initial set, we identified only one mutant line that behaved differently from Col-0 wild-type plants in that its flowering time did not respond to the N treatment. A mutation in the *SOC1* locus caused ON and LN plants to flower at the same time in both LD and SD conditions (Fig. 2a; Table S2). This observation indicates that nitrate-dependent flowering requires a functional SOC1. Interestingly, flowering data from Castro Marin *et al.* (2011) had already indicated that the *soc1-1* mutant (*Ler* background) grown on agar plates displays a reduced response to nitrate. However, as the wild-type *Ler* accession itself presented only a weak response to nitrate in their conditions, the authors did not conclude from their results that SOC1 contributes to nitrate-dependent regulation of flowering.

SOC1 serves as a central integrator for multiple flowering pathways in the SAM (Srikanth & Schmid, 2011) and its expression is strongly upregulated in the center of the meristems when plants are shifted from noninductive SD conditions to inductive photoperiods (LD). To further examine the role of SOC1 in nitrate-dependent flowering, we grew plants in ON and LN under SD conditions for 30 d and then shifted them to LD conditions and analyzed the expression of *SOC1* at the SAM by qRT-PCR analysis and RNA *in situ* hybridization using *SOC1* as a probe (Fig. 2b,c). *SOC1* expression was upregulated in response to an inductive photoperiod at a much later time point and had greatly reduced levels in LN plants, indicating that SOC1 is an essential player in nitrate-dependent flowering at the SAM.

These findings contradict with an earlier study carried out using an agar growth system (Liu *et al.*, 2013). Here the authors demonstrated that low nitrate concentrations (1 mM nitrate and 4 mM glutamine, agar-based) induce the expression of *SOC1* in rosette samples at a 10-leaf stage in a 12 h : 12 h light : dark photoperiod, and suggested that this induction is mediated by an increase in the biosynthesis of GA, causing an early-flowering phenotype (Liu *et al.*, 2013). In addition, another study (1 mM vs 3 mM KNO₃, vermiculite- or agar-based) suggested that GA signaling is involved in the late-flowering phenotype caused by high nitrate content (Gras *et al.*, 2018). In order to discover

whether the GA pathway is affected, five transcripts encoding components of the GA biosynthesis and its signaling were analyzed in SD conditions. We did not detect any differences between ON and LN plants (Fig. S4), demonstrating that nitrate does not interact with GA biosynthesis and signaling when plants have the chance to adapt to available nitrate conditions in soil.

Nitrogen and C are the most important elements to establish normal plant growth and development, and an intricate crosstalk between their signaling networks exists to maintain a balance between N and C metabolism (Nunes-Nesi *et al.*, 2010). C in the form of sucrose is translated to the flowering network and diverse metabolic pathways via the T6P pathway (Wahl *et al.*, 2013; Figueroa *et al.*, 2016), suggesting that it is a hub within an N-/C-sensory checkpoint. The T6P pathway is required for expression of *FT* and affects the age pathway at the level of *SPL3-5* partially via the miR156 (Wahl *et al.*, 2013). Knockdown plants of *TREHALOSE PHOSPHATE SYNTHASE 1* (*TPS1*, 35S::amiRTPS1), which encodes the enzyme producing T6P, still responded to LN (Fig. S5a; Table S2). This indicates that nitrate-dependent flowering acts in parallel with the T6P pathway to regulate flowering time. Further, mutants in *FT*, *TWIN SISTER OF FT* (*TSF*), *CONSTANS* (*CO*), *FLOWERING LOCUS D* (*FD*) and miR156-overexpressing plants showed delayed flowering in LN compared with ON conditions (Fig. S5b-d; Table S2), demonstrating that the photoperiod and the age pathway are not involved in nitrate-dependent flowering. LN also had only a marginal effect on transcript abundances of genes assigned to the photoperiod pathway (Fig. S5e, f) including known floral repressors (e.g. *SMZ*), which have previously been suggested to be involved in the late-flowering phenotype of plants grown in a high-N content growth regime (Gras *et al.*, 2018).

As already mentioned, wild-type plants show a much weaker response to LN in LD than in SD conditions, where the promoting effect of the photoperiod pathway is absent (Fig. 2a; Table S2). Further evidence that the photoperiod pathway is not involved in and may even partly override the LN response is provided by two further observations. First, *ft/tsf* double mutants, which are blocked in the photoperiod response, showed a late-flowering phenotype in LN under LD conditions (± 70 d), which resembled ON-grown wild-type plants under SD conditions (Fig. S5d; Table S2). Second, ON and LN *ft/tsf* plants under LD conditions showed a similar late-flowering response to that of wild-type plants under SD conditions (Figs 2a, S5d).

## Nitrate assimilation at the SAM is regulated by N availability

To date, published data have demonstrated that nitrate after uptake in the roots can be assimilated, stored in vacuoles or transferred to different parts of the plant to foster growth (Tischner, 2001). However, no evidence has come to light on whether nitrate enters and is assimilated in the SAM. If this were to occur, it would open up the possibility that the available nitrate in the SAM can directly regulate flowering genes. In an attempt to understand whether meristematic tissue contains and can assimilate nitrate, we examined the expression of *NITRATE*
REDUCTASE 1 and 2 (NIA1 and NIA2) genes by RNA *in situ* hybridization and qRT-PCR in the SAM (Fig. 2d,e). They encode enzymes catalyzing the first step in nitrate assimilation (Krapp *et al.*, 2014). *NIA1* and *NIA2* are nitrate-induced genes and belong to the primary nitrate response, with changes occurring within 30 min. This first wave of the transcriptional response to nitrate does not require *de novo* protein synthesis or the presence of nitrate reductase, showing that these transcripts respond directly to nitrate (Gowri *et al.*, 1992). Using transcript-specific probes for *NIA1* and *NIA2* on longitudinal sections of the apex, we found that both genes are expressed in the SAM (Figs 2d, S6a, b). Transcript of *NIA1* was detected in leaves, the borders of the rib (RZ) and peripheral (PZ) zones of the SAM and the axillary meristem, whereas the transcript of *NIA2* was detected in the center of the SAM, stretching from L3 into the RZ, and no transcript was detected in the PZ. Most importantly, using both RNA *in situ* hybridization and qRT-PCR, we found that expression of both genes was greatly reduced in LN compared with ON plants (Figs 2d,e, S6a,b), indicating that nitrate is regulating *NIA* expression in the SAM of these plants. This was corroborated by activity measurements in excised apices grown in SD conditions for 30 d (Fig. 2f) and by measurements of nitrate, which revealed significantly reduced concentrations in the SAM of LN vs ON plants (Fig. 2g). With these findings we provide the first evidence that nitrate metabolism can take place in the SAM proper and that nitrate-dependent signaling might directly interact with processes at the SAM.

Nitrate-dependent regulation of *SPL* gene expression at the
SAM

Sequence analysis in several plant species has identified a con-
served motif, termed the nitrate-responsive *cis*-element (NRE), in
the upstream regions of many nitrate-induced genes (Konishi &
Yanagisawa, 2010). The NRE sequence has been reported to be
both necessary and sufficient for nitrate induction (Konishi &
Yanagisawa, 2011). In addition, Konishi & Yanagisawa (2011)
found that NRE-dependent transcription is only activated by
nitrate and not by other N sources. We analyzed upstream inter-
genic regions of core players of the flowering network for the
existence of putative NRE motifs (Table S4) and identified such
motifs predominantly in *SPL3*, *SPL4* and *SPL5* (Fig. 3a;
Table S3), which are expressed at the SAM during the floral tran-
sition (Schmid *et al.*, 2003). NRE motifs were not found in the
upstream intergenic regions of other *SPLs*, such as *SPL9* or
*SPL15*, which are also described to control flowering (Hyun
*et al.*, 2016). We generated transgenic plants harboring synthetic
promoters with four copies of the respective NRE motif fused to
a 35S minimal promoter driving the *GUS* reporter gene (Konishi
& Yanagisawa, 2010) (Fig. 3b). Histochemical staining of
seedlings grown for 4 d on full nutrition ½ Murashige & Skoog
medium (10.3 mM NH₄NO₃, 9.4 mM KNO₃) resulted in
strong stains for the positive control (NRE*NIR1*) as well as
for NRE*SPL5-1* and NRE*SPL5-3*, weaker stains for NRE*SPL3-1*,
NRE*SPL3-2*, NRE*SPL5-2* and NRE*SPL5-4*, and no stain for
NRE*SPL4-1* and the negative control (Fig. 3c). These results

demonstrate that the NRE motifs present in the upstream inter-
genic regions of *SPL3* and *SPL5* can activate transcription.

We next analyzed *SPL* expression via RNA *in situ* hybridiza-
tion in the SAM after transferring ON or LN plants from SD to
inductive LD conditions. Compared with ON plants, LN plants
showed a delay in the rise of *SPL3* and *SPL5*, but not *SPL4* tran-
script (Fig. 3d). Also, under continuous SD conditions the rise in
expression of *SPL5* at the SAM was delayed in LN vs ON plants
(Fig. 3e). qRT-PCR analyses on apices confirmed this result
(Fig. 3f), indicating that timely expression of *SPL3* and *SPL5* at
the SAM requires a positive input by nitrate signaling. In addi-
tion, *SPL4* expression was reduced at the SAM of LN plants
(Fig. 3f), suggesting a tissue-specific, but presumably NRE-
independent, effect of N supply on the regulation of *SPL4*.

NIN-LIKE PROTEIN transcription factors bind to NREs in the presence of nitrate via their RWP-RK DNA-binding domain to affect expression of downstream nitrate-responsive genes (Krapp *et al.*, 2014). NLP7 is known as the master regulator of nitrate signaling and is controlled at a subcellular level by a nitrate-dependent nuclear retention mechanism (Marchive *et al.*, 2013). NLP6 function is described as partially redundant to NLP7 (Guan *et al.*, 2017). A late-flowering phenotype has been observed for the *nlp7* mutants (Castaings *et al.*, 2009). When we grew *nlp6* and *nlp7* mutant plant lines on full-nutrition soil in LD and SD conditions, we found that, for both mutants, flowering time was delayed compared with wild-type plants, whereas the double *nlp6 nlp7* mutant flowered significantly later than either of the single mutant lines (Fig. 4a; Table S2). Furthermore, using RNA *in situ* hybridization, we detected *NLP6* and *NLP7* expression at the SAM (Fig. 4b). These results indicate that both NLPs are candidates for the direct regulation of *SPL3* and *SPL5* via the NREs in their promoters, suggesting a novel mechanism by which the nitrate content in the SAM acts directly to regulate expression of flowering time genes and promote flowering.

Flowering time in LN plants under SD conditions depends
on the T6P pathway

As mentioned before, in LD conditions the photoperiod pathway
may partly override the negative effect of LN and lead to flower-
ing, albeit with a delay. LN plants also eventually initiated flow-
ering in SD conditions, when the photoperiod pathway was
inactive (Fig. 5c). This implies that in SD conditions a further
flower-inducing signal overrides low N. We noticed that sucrose
and T6P concentrations increased in rosettes of LN wild-type
plants grown under SD conditions towards the end of the growth
phase (Fig. 5a). This finding is consistent with an earlier study
showing that nitrate starvation affects T6P concentrations in a
liquid culture with seedlings (Yadav *et al.*, 2014). To test whether
this rise in T6P overrides the effect of low N, we grew 35S::
*amiRTPS1* plants in LN under SD conditions. T6P concentra-
tions increased in LN-grown 35S::*amiRTPS1* plants (Fig. 5b) but
the rise was delayed and T6P concentrations remained below
wild-type concentrations. Strikingly, the 35S::*amiRTPS1* plants
never flowered (Fig. 5c; Table S2). We previously reported that
constitutive expression of *MIR156b* in the background of 35S::
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Nitrate-dependent expression of SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes at the shoot apical meristem (SAM) of *Arabidopsis thaliana*. (a) Putative nitrate-responsive elements (NREs) in the upstream regulatory regions of *SPL3*, *SPL4* and *SPL5*. (b) Schematic illustration of expression cassettes consisting of four copies of the respective NREs fused to a 35S minimal promoter (minP) driving a *GUS* reporter gene and negative control without NRE. (c) Histological staining of the synthetic promoter-*GUS* lines compared with the negative control. (d) RNA *in situ* hybridization using specific probes for *SPL3*, *SPL4* and *SPL5* on longitudinal sections through apices of plants grown in the two nitrogen (N) regimes (optimal N soil (ON) and limited N soil (LN)) in short-day (SD) conditions for 30 d, before shifting them to long days (LD) for 3, 5 and 7 d (DAS, days after the shift). Bar, 100 µm. (e) RNA *in situ* hybridization using a specific probe for *SPL5* on longitudinal sections through apices of plants grown in continuous SD conditions and harvested at the end of the day. (f) Transcript abundances of *SPL3*, *SPL4* and *SPL5* measured by quantitative reverse transcription polymerase change reaction in apices. Bars: (c) 1 mm; (d) 100 µm; (e) 50 µm. Error bars denote SD; the statistical significance between ON and LN was calculated using Student's *t*-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Master regulators of nitrate signaling are present at the shoot apical meristem (SAM) of *Arabidopsis thaliana*. (a) Flowering time analyses of plants mutant for *NIN-LIKE PROTEIN 6* (NLP6) and *NLP7* based on 'time to bolting' (d), determined in long-day (LD) and short-day (SD) conditions on standard full-nutrition soil. (b) RNA *in situ* hybridization on longitudinal sections through inflorescence apices of plants grown in LD conditions using specific probes against NLP6 and NLP7. Error bars denote SD; the statistical significance between optimal N soil (ON) and limited N soil (LN) was calculated using Student's *t*-test: ***, *P* < 0.001.</figcaption></figure>

*amiRTPS1* plants yields plants that are unable to flower on standard soil in SD conditions (Wahl *et al.*, 2013). Growth in LN leads to decreased expression of the *SPLs* in the SAM by limiting nitrate (Fig. 3d–f). It is likely that the nonflowering phenotype of LN-grown *35S::amiRTPS1* plants in SD conditions is due to a stalled T6P pathway in combination with decreased *SPL* expression. Taken together, our data demonstrate that floral induction in the late-flowering wild-type plants grown under nitrate-limited and SD conditions largely relies on the T6P pathway. If both pathways are impeded, flowering cannot occur, highlighting the joint importance of the nitrate signaling pathway and the T6P pathway for the onset of flowering.

Discussion

Understanding the molecular mechanisms underlying the regula-
tion of nitrate-dependent flowering is a crucial step towards the
development of alternative breeding strategies for a sustainable
production of staple crops under nitrate-limited conditions. In
contrast to the growth systems used in previous studies (reviewed
in Lin & Tsay, 2017), we used a soil-based low-N system
(Tschoep et al., 2009) in which plants are able to adapt their
metabolism and growth to the reduced N supply. This allowed us
to study the control of flowering time without complications due
to stress or major changes in central metabolite concentrations.

LN prolongs the adult vegetative phase and delays flowering of *A. thaliana*, but the length of the juvenile vegetative phase is not altered. As seen previously, expression of some of the miR156 precursors are increased in LN leaves (Pant *et al.*, 2009; Krapp *et al.*, 2011; Liang *et al.*, 2012), although expression levels of miR156 targets do not change in response to LN, contradicting earlier assumptions (Vidal *et al.*, 2014). However, we cannot exclude the possibility that these changes become relevant at more extreme nitrate conditions, as predicted by Lin & Tsay (2017).

We found that none of the flowering pathways originating in
leaves caused the delayed flowering phenotype in LN. This
includes all members of the photoperiod pathway as well as its
repressors, although these have recently been associated with high
N-dependent flowering (Gras et al., 2018). The SAM produces
all of the aerial organs of a plant and, as such, major changes
related to the floral transition, that is the production of flowers
instead of leaves, are realized at the SAM. However, some of the
signals that control this transition are initiated in leaves (Srikanth
& Schmid, 2011). Indications that nitrate directly acts as a signal
for flowering were already found in previous studies in which
supplementation of a limited nitrate medium with glutamine did
not rescue the flowering phenotype (Castro Marin et al., 2011;
Weber & Burow, 2018). However, evidence that nitrate signaling
can directly interact with the flowering network at the SAM has
not yet been reported. A recent publication describes a
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Nitrate-signaling and the trehalose 6-phosphate (T6P) pathway act independently to control flowering in *Arabidopsis thaliana*. (a–c) Sucrose and T6P concentrations were measured in rosettes of wild-type plants grown in the two nitrogen (N) regimes in short-day conditions (SD) and harvested at the end of the day (a, b). Sucrose and T6P (a) concentrations rose in plants grown in limited N soil (LN plants), whereas the concentrations stayed constant in plants grown in optimal N soil (ON plants) throughout the experiment and only appeared to be significantly changed in wild-type plants in very old, senescing plant material (compare dark gray columns and 60 vs 80 d after germination (DAG) in panel b). As previously reported, sucrose and T6P concentrations are higher and lower (b), respectively, in rosettes of nonflowering 35S::amiRTPS1 line grown in LN (c) (Wahl et al., 2013). (d) Hypothetical graph on the species-specific relationship between N abundance and its effect on plant growth and flowering time in plants. Error bars denote SD; the statistical significance was calculated using Student's t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.</figcaption></figure>

root-borne cytokinin signal that transduces nitrate availability to the SAM within a matter of days and controls the stem cell population and hence meristem size and growth (Landrein et al., 2018). It remains an open question whether this systemic signal also contributes to the regulation of flowering as previously suggested (D'Aloia et al., 2011). Our findings demonstrate that nitrate does not necessarily need a second messenger to transduce its status to the flowering network in the SAM. The local sensing of nitrate at the SAM, as strongly suggested by our study, will allow changes in the meristematic nitrate concentration to be rapidly translated into downstream events, allowing high developmental plasticity in fluctuating environmental conditions.

We found that nitrate reaches the SAM, where it can be assimilated, as also demonstrated by the presence of *NIA1* and *NIA2*

transcripts and nitrate reductase activity measured in excised apices. These genes, which are part of the primary transcriptional response to nitrate, are repressed in the SAM under limited nitrate availability. We also found that NLP transcription factors, which convey nitrate content information to important downstream targets within the flowering network, are present in the SAM. Both *SPL3* and *SPL5* carry two and five functional NRE sequences in their upstream intergenic regions, respectively. These elements are targeted by NLP6 and NLP7 upon activation and nuclear retention in the presence of nitrate (Marche *et al.*, 2013; Guan *et al.*, 2017). NLP6 and NLP7 belong to a family of nine nitrate-regulated transcription factors present in the *A. thaliana* genome, and all nine members can potentially bind to NREs (Konishi & Yanagisawa, 2013). Currently, NLP7 is
considered the master regulator for nitrate signaling, conveying
information of a cell's nitrate status to metabolic and develop-
mental processes (Castaings *et al.*, 2009). However, other NLPs
have also been described to function in distinct developmental
processes (Yu *et al.*, 2016). Given that they are expressed in
almost all plant organs (Winter *et al.*, 2007; Castaings *et al.*
2009; Chardin *et al.*, 2014), it will be interesting to uncover the
functional significance of each of the NLPs in the future.
Although we did not identify a functional NRE in *SPL4* in the
seedling GUS assay, its expression is decreased at the inflores-
cence SAM of LN plants, suggesting a different signaling mecha-
nism. For the time being we also cannot exclude an additional
contribution of miR156, as at least *MIR156A* and *MIR156C*
levels were slightly but significantly increased in leaves of LN
plants at some times in our analyses. Interestingly, none of the
observed changes in leaves or the SAM led to a prolongation of
the juvenile vegetative phase.

Two lines of evidence support the idea that a functional SOC1, a central integrator for several flowering pathways at the SAM, is required for the nitrate response; first, soc1-6 mutant plants flower at the same time in both growth regimes and, second, SOC1 expression is severely reduced in wild-type plants under nitrate-limited conditions. SOC1 transcript increases in 35S::MIM156 plants, arguing for an effect of the age pathway on SOC1 expression at the SAM (J. W. Wang et al., 2009). In addition, the SOC1 genomic region contains several GATC boxes, which are known target sequences of SPLs, and overexpression of a miR156-resistant version of SPL3 led to an increased expression of SOC1. We therefore postulate an indirect nitrate effect on SOC1 via transcriptional regulation of SPL3 and SPL5 through NLP6 and NLP7. Interestingly, SOC1 was also shown to feed back on SPL3-5 independently of miR156 by directly interacting with their CArG boxes (Jung et al., 2016).

In addition to the NRE motifs present in the upstream
intergenic regions of *SPL3* and *SPL5*, we found more poten-
tially interesting NRE motifs in 5' intergenic regions of other
flowering time genes (e.g. *SMZ*, *TOE2*, *TOE3* and *MIR156F*).
However, no significant or relevant changes in expression of
those genes were observed in the conditions used in our study,
which is why we did not pursue them further for functional
relevance. The plants grown on limited N in our study did
not suffer from acute nitrate starvation. This may explain why
we did not find some of the targets reported in studies that
used more severe conditions. We assume that changes of some
of these genes, in particular regulators of *FT*, become relevant
only when N supply is more strongly reduced and triggers a
general stress level, that is, conditions in which flowering is
reported to be induced rather than delayed relative to ON.
For example, in a recent study (Gras *et al.*, 2018), *SMZ* and
its close homolog *SNZ* were positively regulated by nitrate and
this was associated with delayed flowering in high N. However,
their data indicated they are induced by an indirect route via
the GA pathway (Gras *et al.*, 2018), which did not change in
our growth system. We believe that the nine NLPs encoded by
the *A. thaliana* genome add great flexibility for tissue and

affinity specificity for the individual NRE motifs. Low affinity
for NRE motifs might become relevant, for example, when
nitrate rises to concentrations that exceed those to which plants
can easily adapt, leading to growth retardation, including a
severe delay in flowering.

Interestingly, we found that the nitrate and T6P pathways con-
verge at the same node within the flowering network. In addition
to induction of *FT* in leaves, the T6P pathway activates *SPL3-5*
expression in the SAM, acting partly via a miR156-dependent
pathway and partly via a miR156-independent pathway (Wahl
et al., 2013). To date we cannot tell which of the *SPLs* is more
sensitive to nitrate or T6P. It will also be interesting to learn
whether the T6P and nitrate pathways converge at other impor-
tant checkpoints as well, such as shown for yeast (Wilson et al.,
2007) and plants (Figueroa et al., 2016) where post-translational
regulation of nitrate reductase strongly depends on the T6P sig-
naling pathway. In this sense, residual TPS1 and T6P in the
35S::amiRTPS1 knockdown line might still be able to induce
flowering in ON but no longer suffices to induce flowering in
LN.

Our data reveal the crucial role of central players of the
flowering time network at the SAM in the perception of soil
N concentrations, which can only be overridden by an input
of the T6P pathway in SD conditions or the photoperiod
pathway in LD conditions. This highlights the importance of
these pathways in the regulation of flowering time in response
to the metabolic state of the plants. Taking this as a basis, new
strategies for improving crop performance under nitrate-limited
conditions are possible.

Acknowledgements

We thank M. Stitt, F. Kragler, K. Köhl and the Wahl group for
discussions; S. Prat, M. Schmid and M. Stitt for critical reading
of the manuscript; M. Schmid for seeds and plasmids; J. Kaptur
for subcloning NRE constructs; V. Gramma for initial GUS
stainings; B. Encke, M. Höhne and N. Krohn for measuring
metabolites; and Y. Texier for producing the *nlp7-1 × nlp6-2*
double mutant. Work in the Wahl group is supported by the
BMBF (031B0191), the DFG (SPP1530: WA3639/1-2, 2-1)
and the Max-Planck-Society. Work at the IJPB benefits from the
support of the LabEx Saclay Plant Sciences-SPS (ANR-10-
LABX-0040-SPS and ANR14-CE19-0008, IMANA) to AK.

Author contributions

VW and AS conceived and designed the experiments. JJO, AS
and VW performed all experiments, except for the main part of
the plant work, generation of the NRE lines and the GUS stain-
ings (CA), gathering of RNA *in situ* hybridization and flowering
time data for NLPs (JvD), measurements of T6P concentrations
(RF), analysis of the vegetative phase change and growth parame-
ters (MAD), and generating and verifying *nlp* mutant lines (AK).
JJO, AS and VW analyzed the data. VW wrote the paper with
contributions from JJO and AS. All authors read and commented
on the manuscript before submission.
ORCID

Christin Abel https://orcid.org/0000-0002-8608-0763

Magdalena Anna Działo https://orcid.org/0000-0003-2846-2074

Regina Feil https://orcid.org/0000-0002-9936-1337

Anne Krapp https://orcid.org/0000-0003-2034-5615

Justyna Jadwiga Olas https://orcid.org/0000-0002-4311-6738

Armin Schlereth https://orcid.org/0000-0001-9383-2145

Judith Van Dingenen https://orcid.org/0000-0001-7887-6000

Vanessa Wahl https://orcid.org/0000-0001-7421-8801

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## Supporting Information

Additional Supporting Information may be found online in the Supporting Information section at the end of the article.

**Fig. S1** N-limited *Arabidopsis thaliana* wild-type (Col-0) plants display developmental changes but no response to stress.

**Fig. S2** Leaf initiation rate *Arabidopsis thaliana* (Col-0) plants.

**Fig. S3** The vegetative phase transition is not changed in nitrate-limited *Arabidopsis thaliana* plants.

**Fig. S4** Expression analyses of relevant genes in gibberelic acid (GA) signaling in *Arabidopsis thaliana*.

**Fig. S5** Analyses of the components of the trehalose 6-phosphate (T6P), age and photoperiod pathways in *Arabidopsis thaliana*.

**Fig. S6** Expression analyses of nitrate assimilation genes at the shoot apical meristem (SAM) in *Arabidopsis thaliana*.

**Table S1** Oligonucleotides used in this study.

**Table S2** Flowering time data of experiments described in this study.

**Table S3** Analyses of upstream intergenic regions of selected flowering time gene loci.

**Table S4** List of genes associated with the regulation of flowering time analyzed for Table S3.

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the *New Phytologist* Central Office.

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