# LPCAT1 controls phosphate homeostasis in a zinc-dependent manner.

Mushtak Kisko, Nadia Bouain, Alaeddine Safi, Anna Medici, Robert C Akkers, David Secco, Gilles Fouret, Gabriel Krouk, Mark Gm Aarts, Wolfgang Busch, et al.

► To cite this version:

Mushtak Kisko, Nadia Bouain, Alaeddine Safi, Anna Medici, Robert C Akkers, et al. LPCAT1 controls phosphate homeostasis in a zinc-dependent manner. eLife, 2018, 7, 10.7554/eLife.32077. hal-01716614

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1 LPCAT1 controls phosphate homeostasis in a zinc-dependent manner.

3 Mushtak Kisko¹, Nadia Bouain¹, Alaeddine Safi¹, Anna Medici¹, Robert C. Akkers², David Secco¹, 4 Gilles Fouret³, Gabriel Krouk¹, Mark G.M. Aarts², Wolfgang Busch⁴,⁵, Hatem Rouached¹*

6 1- BPMP, INRA, CNRS, Montpellier SupAgro, Univ Montpellier, Montpellier, France.

8 2- Laboratory of Genetics, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.

10 3- Unité Mixte de Recherche 866, INRA, Place Viala, 34060 Montpellier, France.

11 4- Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria

13 5- Salk Institute for Biological Studies, Plant Molecular and Cellular Biology Laboratory, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA

15 † Present address: Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, CA 94305, USA

18 *To whom correspondence should be addressed to:

20 Hatem **ROUACHED**

21 UMR Biochimie & Physiologie Moléculaire des Plantes.

22 INRA- CNRS-SUPAGRO-UM, Cedex 2

23 Montpellier, 34060 France

24 hatem.rouached@inra.fr or hrouached@carniescience.edu

25 Phone: +33 (0) 4 99 61 31 54

26
27 Abstract

28 All living organisms require a variety of essential elements for their basic biological functions.

29 While the homeostasis of nutrients is highly intertwined, the molecular and genetic

30 mechanisms of these dependencies remains poorly understood. Here, we report a discovery

31 of a molecular pathway that control phosphate (Pi) accumulation plants in Zn deficiency. Using

32 genome-wide association studies we first identified allelic variation of the *Lyso-*

33 *PhosphatidylCholine (PC) AcylTransferase 1 (LPCAT1)* gene as the key determinant of shoot

34 Pi accumulation under Zn deficiency. We then show that regulatory variation at the LPCAT1

35 locus contributes significantly to this natural variation and we further demonstrate that the

36 regulation of *LPCAT1* expression involves bZIP23 TF, for which we identified a new binding

37 site sequence. Finally, we show that in Zn deficient conditions loss of function of *LPCAT1*

38 increases the phospholipid Lyso-PhosphatidylCholine/PhosphatidylCholine ratio, the

39 expression of the Pi transporter PHT1;1, and that this leads to shoot Pi accumulation.
## Introduction

All living organisms require an adequate supply of nutrients for growth and survival. Nutrient deficiencies lead to decreased plant survival and lower nutritional value of foods, which has a profound impact on human health (Myers et al., 2014). In particular zinc (Zn) and iron (Fe) deficiencies affect up to 2 billion people worldwide (Hilty et al., 2010). According to the World Health Organization, about 800,000 child deaths per year are attributable to Zn deficiency alone (Akhtar, 2013). The widespread occurrence of deficiencies in micronutrients such as Zn and Fe in human populations is due to low dietary intake (Rouached, 2013; Myers et al., 2014; Shahzad et al., 2014). In the light of crop optimization for yield and nutritional quality, it is therefore an important goal to understand the genetic and molecular basis of plant nutrition. A complicating circumstance is that plant uptake, storage and use of these nutrients are partly dependent of each other (Rouached and Rhee, 2017). For instance, physiological Zn deficiency leads to over-accumulation of phosphorus (P) in the shoots (for review, Bouain et al., 2014; Kisko et al., 2015). Note worthy, when the Zn supply is low, increasing P supply causes a reduction of plant height, delayed development and severe leaf symptoms including chlorosis and necrosis (Ova et al., 2015). At high P supplies, Zn deficiency associated with elevated shoot P levels causes P toxicity (Marschner, 2012). Interestingly, this P-Zn interaction is also recognized in a wide variety of other biological systems, including rats (Wallwork et al., 1983), human cells (Sandström and Lönnerdal, 1989), and multiple fungal species (Freimoser et al., 2006). In *Saccharomyces cerevisiae* yeast, the Zn status acts as a major determinant of the ability to store P(Simm et al., 2007). Much like Zn nutrition, P homeostasis is of global relevance as current agricultural practices require large amounts of P. At the same time, worldwide P reserves are becoming increasingly scarce and a potential P crisis looms for agriculture at the end of this 21st century (Abelson, 1999; Neset and Cordell, 2012). How P and Zn homeostases are coordinated is therefore not only a fundamental biological question but has also serious implications for global agronomic and biotechnological applications.
P is a critical component of many metabolites and macromolecules, including nucleic acids and phospholipids (PLs) (Poirier and Bucher, 2002; Rouached et al., 2010). Of equal importance, Zn provides chemical, structural and regulatory functions in biological systems (Christianson, 1991), for instance as cofactor for hundreds of enzymes, or by binding to PLs to maintain membrane structure (Binder et al., 2001; Sinclair and Krämer, 2012). Plants have evolved the ability to adjust to large fluctuations in external P or Zn supply. P is taken up by the root system in the form of inorganic phosphate (Pi). In *Arabidopsis thaliana* (Arabidopsis), this uptake relies on members of the high affinity Pi transporter family (PHT1) (Nussaume et al., 2011), of which PHT1;1 is the major contributor (Ayadi et al., 2015). Upon P deficiency, the expression of some *PHT1* transporters increases as a result of the activation of the "PHR1-miR399-PHO2" signalling pathway (Bari et al., 2006; Lin et al., 2008; Pant et al., 2008), causing a strong increase in the acquisition of Pi and its subsequent translocation to the shoots (Lin et al., 2008; Pant et al., 2008). In contrast to our understanding of the molecular mechanisms involved in sensing and signalling of Pi abundance (Chiou and Lin, 2011; Zhang et al., 2014), little is known about how plants sense and signal Zn deficiency. A putative working model of Zn deficiency signalling was proposed by (Assunção et al., 2013), which is centred around two essential members of the bZIP transcription factor (TF) family in Arabidopsis, bZIP19 and bZIP23, without which plants are unable to respond to Zn starvation by inducing the expression of genes involved in Zn uptake and distribution such as the zinc transporter ZIP4 (Assunção et al., 2010). Beyond common set of genes targeted by these two TFs, each TF could regulate distinct genes (Inaba et al., 2015), but the identity of distinctive biding site recognized by each remains poorly unknown. Identifying such biding motif is necessary to better understand how plants regulate Zn homeostasis.

The interaction between Zn and Pi homeostasis in plants is also obvious at the molecular level (for reviews, Bouain et al., 2014; Kisko et al., 2015). For instance, Zn deprivation causes an up-regulation of *PHT1;1* and consequently an over-accumulation of Pi in *Arabidopsis thaliana* (Jain et al., 2013; Khan et al., 2014). The expression of Pi uptake transporters is normally
tightly controlled in roots in response to the P status of the plant, but it is clear that this tight control is lost under Zn deficiency. Remarkably, although the involvement of PHOSPHATE RESPONSE1 transcription factor (PHR1) in the coordination of Pi-Zn homeostasis has been demonstrated (Khan et al., 2014), the Zn deficiency-induced Pi uptake transporter expression is independent of the aforementioned canonical "PHR1-miR399-PHO2" signalling pathway (Khan et al., 2014), indicative of room for new discoveries in Pi homeostasis under Zn deficiency in plants.

In this study we set out to identify the genes controlling such novel mechanisms to cause Pi accumulation in shoots of Zn-deficient Arabidopsis plants. Genome wide association (GWA) mapping was employed using a subset of 223 Arabidopsis accessions from the RegMap panel (Horton et al., 2012), which enabled us to demonstrate that there is heritable natural variation of Pi accumulation in responses to Zn deficiency and that one major locus governing this is the *LysoPhosphatidylCholine AcylTransferase 1 (LPCAT1)* gene. Under Zn deficiency, *lpcat1* mutants showed an alteration in the phospholipids *Lyso-PhosphatidylCholine/PhosphatidylCholine* (Lyso-PC/PC) ratio, and an up-regulation of the expression of the main high affinity Pi transporter gene *PHT1;1*. Finally, we demonstrate that *LPCAT1* acts downstream of one of the two key Zn starvation signalling TFs, bZIP23, for which we identified a new binding site sequence. Overall, this study uncovered a novel pathway, in which *LPCAT1* plays a key role in the coordination of Pi homeostasis and Zn deficiency response in plants through modulation of phospholipid metabolism and Pi transporter expression.

## Results

**GWAS identify two candidate genes involved in the accumulation of Pi in the shoot under Zn deficiency.**

To identify genes regulating shoot Pi concentration under Zn deficiency, genome wide association studies (GWAS) were conducted. To do so, a diverse set of 223 Arabidopsis
accessions, selected from the RegMap panel (Horton et al., 2012) was grown on agar medium supplemented with (+Zn) or without Zn (–Zn) for 18 days, before assessing their shoot Pi concentration (Supplementary file 1). As expected, Zn deficiency in shoots of Col-0 plants was associated with the induction of the expression of two Zn-deficiency marker genes, *ZIP4* and *ZIP12* (Jain et al., 2013) (Figure 1-figure supplement 1). Under the +Zn condition, shoot Pi concentration varied across the 223 accessions from 3 - 10 µmol of Pi per gram of fresh weight (median ~ 5.45 µmol · gram⁻¹ fresh weight of Pi) (Figure 1-figure supplement 2A) while in -Zn, it increased to 4 - 16 µmol of Pi per gram of fresh weight (median ~8.23 µmol · gram⁻¹ fresh weight of Pi) (Figure 1-figure supplement 2B). The broad-sense heritability (H²) of the shoot Pi concentrations was 0.63 and 0.47 under +Zn and –Zn conditions, respectively. Using the genotype and the shoot Pi concentration as input, we performed a mixed model (AMM method (Seren et al., 2012)) GWAS that corrects for population structure (Korte et al., 2012) for both Zn conditions (Figure 1B, C, Figure 1-figure supplement 2C-D). Using a non-conservative 10% false discovery threshold (FDR), we identified 13 significant SNPs in 5 distinct genomic loci to be associated with Pi concentration in the shoots, which was specific for the –Zn condition (Figure 1B). The most significantly associated SNP (*P*-value = 5.86*10⁻⁸; FDR 1%) was located on Chromosome 1 (Supplementary file 2). A haplotype analysis centred on the 50-kb region surrounding the significantly associated SNP revealed one main haplotype (depicted in purple) that was associated with the marker SNP and high Pi concentration (Figure 1-figure supplement 3). The significantly associated SNP was located at the upstream and coding regions of two candidate genes, namely *At1g12640* and *At1g12650* (Figure 1D-E). *At1g12650* encodes an unknown protein likely to be involved in mRNA splicing via the spliceosome, and *At1g12640* encodes a member of the *Membrane Bound O-Acyl Transferase (MBOAT)* gene family known as *LysoPhosphatidylCholine AcylTransferase 1 (LPCAT1*, (Wang et al., 2012)). LPCAT1 is an evolutionarily conserved key enzyme that is involved in phospholipid metabolism and more precisely in the Lands cycle (Lands, 1960). In Arabidopsis LPCAT1 has been shown to catalyze the conversion of lysophosphatidylcholine (Lyso-PC) to produce phosphatidylcholine (PC) (Zheng et al., 2012).
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**LPCAT1** is involved in regulating shoot Pi concentration in Zn deficiency.

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In order to determine the causal gene underlying the shoot Pi accumulation Quantitative Trait Locus (QTL) in -Zn, we used a reverse genetic approach. The first thing we studied was to test if any of these two genes is indeed involved in the -Zn specific variation in shoot Pi concentration. Therefore, wild-type Arabidopsis (Columbia-0, Col-0), T-DNA insertion mutant lines for LPCAT1 (At1g12640) (Wang et al., 2012) and for At1g12650 gene were grown for 18 days on +Zn or -Zn media before assessing their shoot Pi concentration. In response to -Zn, Col-0 plants showed a significant increase (~29% increase, P-value < 0.05) in their shoot Pi concentration compared to +Zn conditions (Figure 2A), which is in line with a previous report (Khan et al., 2014). Importantly, while Pi accumulation in response to -Zn in At1g12650 mutants was indistinguishable from Col-0, lpcat1 mutants displayed a significant increase in shoot Pi concentration (~36% increase, P-value < 0.05) (Figure 2A). We confirmed that this increase in shoot Pi concentration in the lpcat1 mutants is specific to the -Zn treatment as no significant differences were observed in the +Zn condition compared to Col-0. These results showed that LPCAT1, and not At1g12650, is involved in regulating shoot Pi concentration in response to Zn deficiency in Arabidopsis. Our further efforts were therefore directed at understanding the transcriptional regulation of LPCAT1 by -Zn, and then at resolving how allelic variation at the LPCAT1 gene contributes to the variation in shoot Pi concentration.

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**LPCAT1** acts downstream of bZIP23 transcription factor.

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To investigate the molecular causational links between Zn/LPCAT1/Pi we analyzed the cis-regulatory elements present within the 1500-bp region upstream of the LPCAT1 start codon (in Col-0 background) using the search tool AthaMap (Bülow et al., 2010). We identified the presence of a single copy of the 10-bp Zinc Deficiency Response Element (ZDRE, RTGTCGACAY)(Assunção et al., 2010), located 377 bp upstream of the ATG (Figure 2B). This motif is a known binding site for the bZIP19 and bZIP23 transcription factors, the key transcriptional regulators of the -Zn response (Assunção et al., 2010). Given the presence of the ZDRE, we hypothesized that the expression of LPCAT1 under -Zn could be controlled by
177 the bZIP19 or bZIP23 TFs. An electrophoretic mobility shift assay (EMSA) was performed,
178 using a 30-bp promoter fragment containing the 10-bp potential ZDRE, which confirmed that
179 both bZIP19 and bZIP23 could bind to this cis-regulatory element (Figure 2C), as had already
180 been shown by (Assunção et al., 2010).

181 Further analysis of the regulatory regions of LPCAT1 led us to identify a new motif
182 GTGTCGAA (5' untranslated region of LPCAT1), very similar to that of the ZDRE motif
183 (RTGTCGACAY) (Figure 2B). Due to the sequence similarity of this newly identified motif to
184 that of ZDRE, we first tested the capacity of bZIP23 or bZIP19 to bind to this motifs.
185 Interestingly, EMSA analysis revealed that bZIP23 could bind to the newly identified motif,
186 while bZIP19 showed an extremely weak (if any) binding capacity to new motif (Figure 2C).

187 These findings strongly support the Zn-dependency of LPCAT1 expression. We therefore
188 determined the transcript abundance of LPCAT1 in shoots of Arabidopsis wild-type plants
189 (Col-0) grown in -Zn for 6, 12 and 18 days. In response to -Zn, transcript accumulation of
190 LPCAT1 was changed, showing significant down-regulation compared +Zn conditions (Figure
191 2D). This result shows that repression of LPCAT1 upon low -Zn is associated with higher Pi
192 levels and suggests that transcriptional regulation of LPCAT1 is important for its involvement
193 in Pi homeostasis. We next tested whether these bZIP TFs could be involved in regulating the
194 expression of LPCAT1 in -Zn. To test this, we determined the expression levels of LPCAT1 in
195 the bzip19 and bzip23 single and bzip19/bzip23 double knock-out mutant lines and WT plants
196 (Col-0) grown for 18 days in +Zn and -Zn conditions. The LPCAT1 transcript was significantly
197 up-regulated in the bzip23 and bzip19/bzip23 mutant lines, compared to Col-0 and bzip19 in -
198 Zn, which showed a significant down-regulation (Figure 2D). This indicates that bZIP23, but
199 not bZIP19, is involved in negatively regulating the expression of LPCAT1 under -Zn. We
200 therefore hypothesized that bZIP23 but not bZIP19 are necessary for the downregulation of
201 LPCAT1 in -Zn and subsequent Pi accumulation and there assessed the capacity of the
202 mutants to accumulate Pi when grown with or without Zn for 18 days. While in +Zn, all plants
203 showed similar shoot Pi content, we observed a significant decrease in shoot Pi content in the
204 bzip23 and bzip19/bzip23 mutants compared to Col-0, confirming the regulatory role of bZIP23
and not bZIP19 (Figure 2E). Taken together, this suggests that bZIP23 represses *LPCAT1* upon -Zn and this repression leads to the over-accumulation of Pi in shoots in Arabidopsis grown under -Zn condition.

**Allelic variation of LPCAT1 determines natural variation of Pi content under zinc deficiency.**

We next wanted to test whether allelic variation of *LPCAT1* is causal for the observed differences in Pi accumulation under -Zn. For this, we selected two contrasting groups of accessions with either a high ratio (Br-0, Ts-1, PHW-2 and Sap-0) or a low ratio (Ang-0, CIBC-5, Col-0, EST-1, RRS-10) of Pi accumulated in shoots of -Zn plants compared to +Zn plants (Figure 3A). Interestingly, comparative sequence analysis of the regulatory regions of *LPCAT1* of these accessions using the sequence data from the 1001 genomes project (Alonso-Blanco et al., 2016) revealed that the common ZDRE motif (Figure 2B) didn't display any variation between these two groups of accession (Figure 3A), and that the newly identified *bzip23* specific motif (Figure 2B) showed clear variation between the two groups of accession with the accessions with low Pi ratio under -Zn exhibiting a Col-0 like *GTGTCGAA* motif and the high Pi accumulating accession displaying a *GTGTCACA* motif (Figure 3A, Figure 3-figure supplement 1, Supplementary file 3). We therefore tested the capacity of bZIP23 and bZIP19 to bind to this latter version (*GTGTCACA*) of the ZDRE motif. EMSA analysis revealed that only bZIP23 could bind to this version of ZDRE motif (Figure 3B). Taken together, EMSA results (Figure 2C, Figure 3B) support the specificity of a new ZDRE motif for bZIP23.

We next assessed the effect of these motif sequence changes on the activity of the *LPCAT1* promoter using a quantitative *in planta* transactivation assay (Bossi et al., 2017). In this assay, we co-transformed tobacco leaves with an effector construct (35S::bZIP23 or 35S::YFP) with a reporter construct, containing either the LPCAT1 Col-0 native promoter (with *GTGTCGAA* motif), the LPCAT1 Col-0 mutated promoter (with *GTGTCACA* motif), or the promoter of the zinc transporter *ZIP4* promoter (as positive control) fused to a *β-glucuronidase* (*GUS*)-encoding reporter gene (Figure 3C). The comparison of the ability of 35S-bZIP23 or 35S-YFP
to activate these *LPCAT1* promoters was performed by quantifying the GUS activity. The average relative activity was calculated as the GUS activity of each promoter in the presence of 35S::bZIP23 divided by its GUS activity in the presence of 35S::C-YFP (Figure 3D). As expected, our results showed an induction of the positive control (ZIP4 promoter by 35S:bZIP23). Consistent with the hypothesis that the natural variation of the new ZDRE is relevant for *LPCAT1* regulation, bZIP23 acted as stronger repressor of the *LPCAT1* Col-0 mutated promoter that contained the new ZDRE of Sap-0 compared to the Col-0 *LPACT1* native promoter (Figure 3D). Moreover, *LPCAT1* was downregulated by a larger extent in accessions that accumulated more Pi upon -Zn (Figure 3E) and contained the new (non Col-0) ZDRE while the expression of *bZIP23* remained unchanged in all accessions and growth conditions tested.

To further test whether the difference in *LPCAT1* expression was due to the natural allelic variation in the regulatory regions and whether this was causal for the Pi accumulation, we focused on only two contrasting accessions, Sap-0 and Col-0, which displayed a significantly different capacity to accumulate shoot Pi in -Zn (Supplementary file 1). Noteworthy, the *LPCAT1* promoter and predicted amino acid coding sequences of Col-0 and Sap-0 displayed 97.9% and 99.4% sequence identity respectively (data not shown). The *lpcat1* knock-out mutant (in Col-0 background) was then transformed with either an empty vector (control) or one of four constructs containing 1.5 kbp of the promoter (immediately upstream of the start codons) of either *pLPCAT1*<sup>Col-0</sup> or *pLPACT1*<sup>Sap-0</sup> respectively fused to either the coding region of *LPCAT1*<sup>Col-0</sup> or *LPCAT1*<sup>Sap-0</sup> (Figure 4A). Three independent, single locus insertion lines (based on segregation of the insertion in progeny of a hemizygous plant) were considered for the analysis. When expressed under the *pLPCAT1*<sup>Col-0</sup> promoter, *LPCAT1*<sup>Col-0</sup> or *LPCAT1*<sup>Sap-0</sup> complemented the *lpcat1-1* knock-out mutant phenotype and showed a similar Pi content to WT (Col-0) plants in both +Zn and -Zn conditions (Figure 4B). This indicates that the polymorphisms in the coding region are not responsible for the change in Pi content in -Zn conditions. In contrast, lines complemented with the *pLPCAT1*<sup>Sap-0</sup>:*LPCAT1*<sup>Col-0</sup> or *pLPCAT1*<sup>Sap-0</sup>:*LPCAT1*<sup>Sap-0</sup> transgenic lines showed significantly higher Pi content compared to *pLPCAT1*<sup>Col-0</sup> lines.
²⁶¹ ⁰:LPCAT1¹Col-⁰ or pLPCAT1¹Col-⁰:LPCAT1¹Sap-⁰ lines or WT (Col-0) in -Zn conditions (Figure 4B).
262 This result demonstrates that regulatory variation in of the LPCAT1 promotor determines Pi
263 accumulation and favours the model that variation in the expression level of LPCAT1 as the
264 cause of the variation in Pi accumulation in -Zn. Therefore, we assessed LPCAT1 mRNA
265 accumulation in in WT (Col-0) and all transgenic lines grown in both +Zn and -Zn conditions.
266 Our result showed that while LPCAT1 is down-regulated in all tested lines by -Zn treatments,
267 the lines complemented with the LPCAT1 driven by pLPCAT1 Sap-⁰ accumulates significantly
268 lower LPCAT1 mRNA than that of those under the control of pLPCAT1 Col-⁰ and WT (Col-0)
269 (Figure 4C). Taken together, our results indicate that the allelic variation between Col-0 and
270 Sap-0 in the promoter of the LPCAT1 gene causes the difference in LPCAT1 expression, and
271 confirm that this difference leads to the difference in Pi accumulation under -Zn. Importantly,
272 the polymorphisms in the bzip23 binding site in the promotor of LPCAT1 suggest a potential
273 cis-regulatory mechanism for this.

274 LPCAT1 mutation impacts phospholipid concentrations in -Zn.

275 While LPCAT1 had not been implicated in any known process involving Zn, it is known to
276 catalyse the conversion of lyso-phosphatidylcholine (Lyso-PC) to phosphatidylcholine (PC) in
277 the remodelling pathway of PC biosynthesis (Figure 5A) (Lands, 1960; Chen et al., 2007;
278 Wang et al., 2012). Consequently, we hypothesized that a mutation in LPCAT1 or bZIP23
279 would affect the Lyso-PC and PC under -Zn conditions. To test this, we measured the
280 composition of these two phospholipid classes in the shoots of the Col-0 wild type and the
281 bzip23 and lpcat1 mutants, in +Zn and -Zn conditions. In +Zn, no significant changes in the
282 Lyso-PC and PC levels in the three different genotypes were observed (Figure 5B, C).
283 However, under -Zn, bzip23 showed a modest (but non-significant) decrease in the Lyso-
284 PC/PC ratio while the mutation in LPCAT1 resulted in a significant increase of Lyso-PC and a
285 decrease of PC, resulting in an increase of the Lyso-PC/PC ratio (~1.2 fold, P-value< 0.05)
286 compared to Col-0 plants (Figure 5D). These results demonstrate that the LPCAT1 function is
287 required to maintain the shoot Lyso-PC/PC ratio under -Zn. We next tested wether the
288 polymorphisms in the regulatory region of *LPCAT1* are responsible for the change in LPC/PC
289 ratio that ultimately affects the Pi content in -Zn conditions. We determined the LPC, PC
290 concentrations in the shoots of the plants expressing *LPCAT1* driven by the LPCAT1<sup>Col-0</sup>
291 promoter (pLPCAT1<sup>Col-0</sup>::*LPCAT1*<sup>Col-0</sup>, pLPCAT1<sup>Col-0</sup>::*LPCAT1*<sup>Sap-0</sup>) or the LPCAT1<sup>Sap-0</sup>
292 promoter (pLPCAT1<sup>Sap-0</sup>::*LPCAT1*<sup>Col-0</sup>, pLPCAT1<sup>Sap-0</sup>::*LPCAT1*<sup>Sap-0</sup>) in the *lpcat1* mutant
293 background, grown in presence or absence of Zn for 18 days. WT plants of the Col-0 and Sap-
294 0 accessions, and *lpcat1* transformed with the empty vector were included in this analysis. In
295 the presence of Zn, no difference in PC or LPCA concentrations was observed between all
296 plant lines. However, under -Zn conditions, Sap-0, pLPCAT1<sup>Sap-0</sup>::*LPCAT1*<sup>Col-0</sup>, pLPCAT1<sup>Sap-</sup>
297 <sup>0</sup>::*LPCAT1*<sup>Sap-0</sup> or *lpcat1* lines showed a significant decrease of PC and increase of LPC
298 concentrations, leading to an increase of Lyso-PC/PC ratios (Figure 6). These results further
299 support the association between the increase of LPC/PC ratios and the alterations in P content
300 in the plant shoots under Zn deficiency (Figure 4b).

301 Accumulation of Pi in *lpcat1* involves the HIGH AFFINITY PHOSPHATE TRANSPORTER
302 *PHT1;1*.

303 While the molecular function of *LPCAT1* is related Lyso-PC/PC homeostasis, it doesn't answer
304 the question how it might cause Pi levels to increase under -Zn conditions. A first hint towards
305 answering this question came from our GWAS data: The 3rd most significant associated peak
306 (8% FDR) under -Zn conditions was located in a region of chromosome 5 containing members
307 of the high affinity Pi transporters *PHT1* gene family, namely *PHT1;1*, *PHT1;2*, *PHT1;3* and
308 *PHT1;6* (Chr5: 17394363 - 174200000) (Figure 7 A-C, Supplementary file 2). Except for
309 *PHT1;6*, the role of these genes in Pi uptake, transport and accumulation in Arabidopsis is well
310 documented (Nussaume et al., 2011; Ayadi et al., 2015). To test, the activity of one of these
311 genes might be related to the *LPCAT1* dependent Pi accumulation under -Zn, we assessed
312 the expression of the *PHT1* transporter genes in the shoots of *lpcat1* mutant and WT (Col-0)
313 plants grown in +Zn or -Zn for 18 days. In all genotypes, *PHT1;1* was the only member of the
314 *PHT1* gene family to be significantly up-regulated in the -Zn condition (Figure 7D). Zn
deficiency induces transcription of *PHT1;1* already ~ 2.2 fold (P<0.05) in WT (Col-0) and this induction was further increased by 2-fold (P<0.01) in *lpcat1* mutants (Figure 7D), when compared to +Zn (Figure 7-figure supplement 1). The expression of the *PHT1;1* was thereafter tested for responsiveness to -Zn in roots of WT (Col-0) and the *lpcat1-1* mutant. While -Zn caused no significant change in expression of the *PHT1;1* in roots of WT, it increased its expression by ~2-fold in roots of *lpcat1* mutant (Figure 7-figure supplement 2). We next determined the effects loss of function for each phosphate transporter located under the second GWAS peak (*PHT1;1*, *PHT1;2* and *PHT1;3*) for the accumulation of Pi in -Zn in 18-day-old plants. The *pht1;1* mutant showed low Pi accumulation in presence of Zn compared to WT plants (Figure 7E) consistently with (Shin et al., 2004) that reported that the *pht1;1* mutant showed a reduction in Pi content of the shoots relative to wild type plants grown under control condition (+Pi+Zn). Importantly, no increase of Pi concentration was observed in the shoots of *pht1;1* grown in -Zn, which contrast with the Pi accumulation in *pht1;2* and *pht1;3* that was in a similar range to WT plants in presence or absence of Zn. These results show the involvement of *PHT1;1* in the overaccumulation of Pi in the shoot of *lpcat1* grown in -Zn, and further supports a second peak of the GWAS on the chromosomal region of *PHT1* genes.

Discussion

Understanding how Zn and Pi homeostasis are wired to regulate growth is crucial to offer a new perspective of improving Pi nutrition in plants by modulating the Zn-deficiency signalling pathway. Our study provides a first insight into the genetic and molecular mechanism that controls shoot Pi concentration under -Zn in plants by discovering a pathway which includes the -Zn response TF *bZIP23* that target the *LPCAT1*, and the Pi transporter *PHT1;1*.

In *A. thaliana*, GWAS has been shown to be a powerful approach to detect loci involved in natural variation of complex traits including variation in the accumulation of non-essentials or toxic elements in plants, such as sodium (Baxter et al., 2010), cadmium (Chao et al., 2012) or arsenic (Chao et al., 2014). Here we used GWAS to identify genes involved in the regulation of
the essential macronutrient (P) concentration in its anionic form (Pi) in plants grown under control conditions (+Zn) and -Zn. In both conditions, our GWA analysis reveals that there is widespread natural variation in shoot Pi concentration, and supports the existence of genetic factors that affect this trait (Figure 1). The GWAS data support the -Zn specificity of this response, since no association was detected around the *LPCAT1* locus in our control condition (+Zn) (Figure 1). The presence of the Zinc Deficiency Response Element (ZDRE) (Assunção et al., 2010) in the promoter of *LPCAT1* and more particularly the newly identified binding motif specific for bZIP23 (Figure 2, 3) in the 5' untranslated leader of *LPCAT1* is a strong argument supporting the Zn-dependency of this response.

A ZDRE is present in the promoter regions of many genes targeted by bZIP19 and bZIP23 (Assunção et al., 2010). In addition to their positive regulatory role by inducing several Zn deficiency related genes, publicly available microarray showed that bZIP19 and bZIP23 may have a negative regulatory role as many genes were induced in the bzip19/bzip23 mutant background compared to WT plants grown in -Zn (Azevedo et al., 2016). A functional redundancy of these two TFs was proposed based on the oversensitivity of the *bzip19* and *bzip23* double mutant to -Zn, which was not observed with either *bzip19* or *bzip23* single mutants (Assunção et al., 2010). This redundancy may not be absolute, as recent physiological and genetic evidence indicates that bZIP19 and bZIP23 are not completely redundant and they not only regulate the same, but also separate sets of genes in Arabidopsis (Inaba et al., 2015). Our results support this finding by showing that only bZIP23 is involved in regulating *LPCAT1* in response to -Zn. bZIP23 is likely to do so through two cis-elements in the non-coding part of the *LPCAT1* gene. One being the aforementioned ZDRE, which can also be bound by the bZIP19 paralogue of bZIP23, the other a novel binding motif, with versions TGTCACA and TGTCGAA, which are specifically bound by bZIP23. Worth noting, in the accessions panel used in our work one other allele can be found, in the Alc-0 accession (TGTCAAA) that displayed the lowest Pi accumulation in our panel of accession (Supplementary file 3, Figure 3-figure supplement 1). Alc-0 is the only accession among all
371 Arabidopsis accessions for which sequence information is available in the 1001 genome database, with this version of the ZDRE motif.
372

373 The new ZDRE motif resides in the 5'-untranslated leader of *LPCAT1*. Binding of bZIP23 to this element therefore might physically block the transcription of the *LPCAT1* gene under Zn deficient conditions. This is further supported by the repressive role for bZIP23 on the expression of *LPCAT1* under Zn deficiency. Genomic sequence surveys screening for this new TF-binding site promise to further help identifying a complete list of genes potentially regulated by bZIP23 in order to fully understand the involvement of bZIP23 in the -Zn response in a genome-wide manner.
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380 Based on our results we propose that the sequence change in the 5'UTR sequence impacts gene(s) expression, and consequently causes variation in the associated traits. It has been proposed that evolution of species complexity from lower organisms to higher organisms is accompanied by an increase in the regulatory complexity of 5'UTRs (Lui et al., 2012).
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384 Nevertheless, so far little is known about the role of the 5'UTR sequence in the regulation of gene expression at the transcriptional level. The role of 5'UTR in the regulation of gene expression is perhaps best studied in human. Several studies showed that point substitutions in the 5'UTR change the expression of several genes such as *Ankirin Repeat Domain 26* (Pippucci et al., 2011), *Solute Carrier Family 2 Member 4* (Malodobra-Mazur et al., 2016), and *Cyclin D1* (Berardi et al., 2003). In contrast, few examples exist on the role of 5'UTR as *cis* regulators in plants. In Arabidopsis, it has been shown that the regulation of *NRP1* gene expression involves WRKY DNA Binding Proteins, which act on a potential *cis*-acting regulatory element located within the 5'UTR of *NRP1* (Yu et al., 2001). More general, a large-scale study of DNA affinity purification sequencing, which is a high-throughput TF binding site discovery method, has revealed a global preference across TF families for enrichment at promoters and 5'UTRs (O'Malley et al., 2016). Taken together, our study has uncovered a new example for the role of the 5'UTR in gene expression regulation and evolution in plants.
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Of a particular interest is that our study revealed that this novel bZIP23-interacting sequence motif is subject to natural variation in *A. thaliana* (Figure 3), and its alteration may be associated with changes in the binding capacity of bZIP23. There are several ways that genetic variants can mechanistically contribute to plant adaptation. Many reported examples with regards to nutrient accumulation involve a change in the coding sequence of a gene that then alters the amino acid sequence of the encoded protein, thus leading to the disruption of gene function and a phenotypic change (Baxter et al., 2010; Chao et al., 2012; Chao et al., 2014). Reports on the role of specific regulatory element polymorphisms in the regulation of complex traits such as nutrient homeostasis crosstalk are less common, also because it is difficult to identify these relevant sequence changes. In our study we demonstrated that allelic variation (SNPs) in the novel bZIP23 binding motif upstream of the *LPCAT1* gene is associated with variation in *LPCAT1* expression levels, which in turn results in variation in Pi accumulation in -Zn conditions. The *LPCAT1* natural variants such as found in this study offer new inspiration for agronomical and biotechnological applications to optimize Pi use efficiency in plants.

While mutation of *LPCAT1* results in altered Lyso-PC and PC concentrations; an altered Lyso-PC/PC ratio; the up-regulation of *LPCAT1* in *bzip23* mutant background has no significant effect on this ratio. The simplest explanation for this observation would be that in the *bzip23* mutant background a portion of the synthesized PC is fed into the Kennedy pathway leading to the biosynthesis of different molecules such diacylglycerol or phosphatidic acid, and therefore no changes in Lyso-PC/PC could be detected (Wang et al., 2012). Nevertheless, an attractive second explanation exists: LPCAT1 could be subjected to a regulation at the protein level as a strategy to optimize phosphatidylcholine specie levels. In animals, it has been proposed that the LPCAT1 primary protein sequence may contain additional motifs, structural features, or interact with second messengers or ligands that can, under certain circumstances, alter its protein half-life (Zou et al., 2011). The precise mechanism that regulates LPCAT1 protein stability by the ubiquitin-proteasomal pathway was already shown (Zou et al., 2011). Further studies will be required to verify the presence of such regulation pathway for LPCAT1 in
426 Arabidopsis, and if confirmed, such a mechanism would add another level of our
427 understanding of LPCAT1 activity in plants.

428 Mutation of *LPCAT1* results in increased *PHT1;1* expression levels (Figure 7D); and ultimately
429 an over-accumulation of Pi under Zn deficiency. The induction of the expression of genes
430 encoding P uptake transporters under Zn deficiency has been reported in crop plants such as
431 barley (*Hordeum vulgare*) (Huang et al., 2000); and the model plant Arabidopsis (Jain et al.,
432 2013; Khan et al., 2014; Pal et al., 2017). Our study showed an induction of *PHT1;1* in *lpcat1*
433 plants grown under Zn deficiency. The increase in *PHT1;1* expression levels is likely to explain
434 the increased shoot Pi concentration in *lpcat1* since it is known that CaMV 35S promoter
435 driven overexpression of this Pi transporter significantly increases shoot Pi concentration
436 (Mitsukawa et al., 1997; Shin et al., 2004; Catarecha et al., 2007). Moreover, our finding
437 provides evidence supporting a role for a Lyso-PC/PC-derived signal in regulating Pi
438 homeostasis under –Zn. Until recently our knowledge on PL-derived signals in plants was
439 scarce; however, physiological and molecular studies have shown that some PL classes could
440 serve as precursors for the generation of diverse signalling molecules (Spector and Yorek,
441 1985; Testerink and Munnik, 2005). For instance, Lyso-PC was shown to act as a signal for
442 the regulation of the expression of arbuscular mycorrhiza (AM)-specific Pi transporter genes in
443 potato, tomato and recently in *Lotus japonicus* (Drissner et al., 2007; Vijayakumar et al., 2016).

444 In addition to the involvement of individual PLs in specific physiological processes in plants
445 (e.g ion transport), a broader importance of changes in Lyso-PC/PC ratio for the regulation of
446 plants development and basic cell biology is emerging. For instance, in Arabidopsis alteration
447 of the Lyso-PC/PC ratio shortens the time to flower (Nakamura et al., 2014). In human cells,
448 the Lyso-PC/PC ratio was also associated with an impairment of cell function, signalling and
449 metabolism (Mulder et al., 2003; Klavins et al., 2015). Our data now demonstrate a
450 fundamental link between PL metabolism, particularly Lyso-PC/PC, and Pi accumulation in –
451 Zn condition, and lays the foundation for exploring the role of Lyso-PC/PC-derived signal in
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controlling ion homeostasis and response to environmental changes not only in plant cells but
also in other organisms.

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Overall, our study shed light on molecular mechanism underlying an old observation made as early as 1970s, namely P-Zn interaction in plants (Warnock, 1970; Marschner and Schropp, 1977; Loneragan et al., 1979). By combining GWAS and functional genomics approaches, we discovered a complete pathway involved in the regulation of shoot Pi accumulation in -Zn that can be defined as bZIP23-LPCAT1(Lyso-PC/PC)-PHT1;1. Beyond its fundamental importance, our study could have a direct impact on plants growth in field by improving plant growth while reducing P supply, and will help meeting one of challenges facing agriculture in the 21st century.

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Materials and Methods

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Plant materials and growth conditions.

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A subset of 223 *Arabidopsis thaliana* accessions of the RegMap panel (Horton et al., 2012) was used for genome-wide association studies (Arabidopsis Biological Resource Center accession number CS77400). The names of accessions are provided in supplementary file 1. All lines were used side by side in the same growth chambers under the same conditions, 22 °C under long days (16 h light and 8 h dark). Arabidopsis mutants used in this study are in the Columbia-0 genetic background. The *bzip19bzip23* mutant previously described by (Assunção et al., 2010) was used in this work. T-DNA insertion mutant lines for the At5g43350 (N666665, *pht1;1*), At5g43360 (N661080, *pht1;2*), At5g43370 (N448417, *pht1;3*), At1g12640 (N686743 (*lpcat1-1*, (Wang et al., 2012)), N442842) and At1g12650 (N526222) genes were obtained from the European Arabidopsis Stock Centre (arabidopsis.info; University of Nottingham, UK). Plants were germinated and grown on vertically positioned agar-solidified media (A1296, Sigma). The complete nutrient medium contained: 9.5 mM KNO3, 10.3 mM NH4NO3, 1.5 mM MgSO4, 1 mM KH2PO4, 2 mM CaCl2, 100 µM FeNaEDTA, 100 µM MnSO4, 30 µM ZnSO4, 100 µM H3BO3, 5 µM KI, 1 µM Na2MoO4, 0.1 µM CuSO4 and 0.1 µM CoCl2 (adapted from
Murashige and Skoog, 1962). Zn-deficient medium was made by omitting ZnSO₄. Seeds sown on plates were stratified at 4 °C for 3 days. Plates were then transferred to a growth chamber for 18 days set at the following conditions: 16/8h light/dark cycle, 250 µmol m⁻² s⁻¹ light, and 24/20 °C (light/dark).

## Plasmid construction and plant transformation.

The *LPCAT1* coding region driven by its native promoter (1.5 kbp fragment immediately upstream of the start codon including the 5' untranslated region (5'-UTR)) from Col-0 and Sap-0 accessions were amplified using PCR and the following primers pLPCAT1<sup>Col-0</sup>-forward 5'-cgctgcaggtgtcgaaaacccgtttt-3'; pLPCAT1<sup>Col-0</sup>-reverse 5'-cgggatcctgatcagagagttacaac aggagag-3'; pLPCAT1<sup>Sap-0</sup>-forward 5'-cgctgcaggtgtcacaacccgggt-3' and pLPCAT1<sup>Sap-0</sup>-reverse 5'-cgggatccatgatcagatagttacaacaggagagg-3', and then cloned into the binary vector pCAMBIA1301 by restriction enzymes BamHI and PstI (site underlined). The *LPCAT1* coding regions were amplified using PCR and the following primers pLPCAT1<sup>Col-0</sup>-forward 5'-cgctgcagttattcttctttacgcggttttg-3'; pLPCAT1<sup>Sap-0</sup>-forward 5'-cgctgcagttattcttctttacgtggttttggt-3' and pLPCAT1<sup>Col-0/ Sap-0</sup>-reverse 5'-cgctgcagatggatatgagttcaatggctg-3'. PstI was used for the fusion of pLPCAT1<sup>Col-0</sup> or pLPCAT1<sup>Sap-0</sup> promoters to either LPCAT1<sup>Col-0</sup> or LPCAT1<sup>Sap-0</sup>. The constructs were transformed into *Agrobacterium tumefaciens* strain GV3101 and then used for Arabidopsis transformation by the floral dip method (Clough and Bent, 1998). Transgenic plants were selected by antibiotic resistance, and only homozygote descendants of hemizygote T2 plants segregating 1:3 for antibiotic resistance: sensitivity were used for analysis.

## Inorganic phosphate concentration measurements and GWA Mapping

All accessions were grown in the presence or absence of zinc for 18 days. Shoots were collected, weighed and ground into powder in liquid nitrogen. An aliquot (30 mg) was incubated at 70 °C in NanoPure water, for 1 hour. Inorganic phosphate (Pi) concentrations were determined using the molybdate assay as previously described by (Ames, 1966).
shoot Pi concentrations across the analysed accessions was used as phenotype for GWA
analysis. The GWA analysis was performed in the GWAPP web interface using the mixed
model algorithm (AMM) that accounts for population structure (Seren et al., 2012) and using
the SNP data from the RegMap panel (Atwell et al., 2010; Brachi et al., 2010; Horton et al.,
2012). Only SNPs with minor allele counts greater or equal to 10 (at least 10 out of 223
accessions contained the minor allele) were taken into account. To correct for multiple testing,
the false discover rate was calculated using the Benjamini-Hochberg correction (Benjamini
and Hochberg, 1995). An FDR threshold of 0.1 was used to detect significant associations.

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## Haplotype analysis

Haplotype analysis was performed as follows. SNPs from a 50 kb window around the
significant marker SNP (chromosome 1, position 4306845) were extracted for the 221 natural
accessions. SNP data was taken from the Regional Mapping Project SNP panel described in
Horton et al. (2012). These SNPs were used as the input for fastPHASE version 1.4.0 (Scheet
and Stephens, 2006). Results were then visualized using R.

525

## Gene expression analysis by quantitative RT-PCR

For expression analysis, the Plant RNeasy extraction kit (Qiagen) was used to extract total
RNA free of residual genomic DNA from 100 mg frozen shoot material. Total RNA was
quantified with a NanoDrop spectrophotometer (Thermo Scientific). Two µg of total RNA was
used to synthesize cDNA. Reverse transcriptase PCR (RT-qPCR) was performed with a Light
Cycler 480 Real-Time PCR System (Roche) using SYBR green dye technology (Roche) as
described by (Khan et al., 2014). The primers used in this study are LPCAT1-forward 5'-
ggtgttaagcttgcacgaaac-3'; LPCAT1-reverse 5'-agagaaacaagaaccgga-3' and UBQ10-forward
5'-aggatggcagaaactcttgct-3'; UBQ10-reverse. 5'-tcccagtcaacgtcttaacg-3'. The primers used to
quantify ZIP4 are ZIP4-forward 5'-cggttaaacataagaaatcaggagc-3'; ZIP4-reverse 5'-
taaatctcgagcgttgtgatg-3'; and for ZIP12 are ZIP12-forward 5'-aacagatctcgcttggcg-3'; ZIP12-
reverse 5'-aatgtgatcatcatcttggg-3'. Primers used to quantify the PHT1 gene family member are
designed according to (Khan et al., 2014). Quantification of mRNA abundance was performed
in a final volume of 20 µL containing 10 µL of the SYBR Green I master mix, 0,3 µmol primers, and 5 µL of a 1:25 cDNA dilution. PCR conditions were as 95°C for 5 min, and followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, 72 °C for 25 s. One final cycle was added in this program: 72 °C for 5 min. For every reaction, the cycle threshold (Ct) value was calculated from the amplification curves. For each gene, the relative amount of calculated mRNA was normalized to the calculated mRNA level of the *Ubiquitin10* control gene (*UBQ10*: At4g05320) and expressed as relative values against wild-type plants grown in the presence or absence of Zn in the medium. Quantification of the relative transcript levels was as described in Rouached et al., 2008). The mRNA abundance of each gene was calculated following normalization against the CT values of *Ubiquitin10* mRNA, for instance ΔCt, *LPCAT1* = Ct, *LPCAT1* – (Ct, *UBQ10*). Quantification of the relative transcript levels was performed as following, low Zn (-Zn) treatment was compared to control (Ct, +Zn), the relative mRNA accumulation of each gene was expressed as a ΔΔCt value calculated as follows: ΔΔCt = ΔCt, *LPCAT1*(-Zn) – ΔCt, *LPCAT1* (Ct). The fold change in relative gene expression was determined as 2⁻⁵⁻⁴ΔΔCt.

553

## Expression and purification of bZIP19 and bZIP23 proteins.

bZIP19 and bZIP23 coding sequences CDS were first cloned in the pENTR/D-TOPO vector, and then transferred to pDEST15 vector (Invitrogen) by LR reaction following the manufacturer's instructions. The GST-bZIP19 and GST-bZIP23 fusion proteins were expressed in *Escherichia coli* Rosetta 2(DE3)pLysS (Novagen, Darmstadt, Germany). Transformed cells were grown in a phosphate-buffered rich medium (Terrific broth) at 37°C containing appropriate antibiotics until the OD₆₆₀ reached 0.7-0.8. After induction with 1 mM IPTG (isopropyl-b-D-thiogalactoside) for 16 h at 22° C, bacteria were harvested by centrifugation (6000 ×g, 10 min, 4°C) and suspended in 1X PBS buffer containing lysozyme from chicken egg white (Sigma) and complete protease inhibitor cocktail (Roche). The resulting cell suspension was sonicated and centrifuged at 15,000 ×g, for 15 min at 4°C to remove intact cells and debris. The proteins extract was mixed with buffered glutathione

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566 sepharose beads (GE Healthcare, Freiburg, Germany), and incubated at 4℃ for 3 h. The resin
567 was centrifuged (500 ×g, 10 min, 4℃) and washed five times with 1X PBS buffer.

568 bZIP19 and bZIP23 were then cleaved from GST using 25 unit/ml of thrombin at room
569 temperature for 16h. All fractions were subjected to SDS-PAGE, and proteins concentrations
570 were determined. For protein quantification, absorbance measurements were recorded on a
571 nanodrop spectrophotometer (Model No.1000, Thermo Scientific Inc., Wilmington, Delaware,
572 USA) at 280 nm, and in parallel on a VICTOR2™ microplate reader (MULTILABEL
573 COUNTER, life sciences) at 660 nm using the Pierce 660 nm Protein Assay (Pierce/Thermo
574 Scientific, Rockford; (Antharavally et al., 2009)).

575

## Electrophoretic Mobility Shift Assay (EMSA)

577 EMSA was performed using purified proteins and DNA probes labeled with Biotin-TEG at the
578 3' end. Biotin-TEG 3' end-labeled single-stranded DNA oligonucleotides were incubated at
579 95 °C for 10 min and then annealed to generate double-stranded DNA probes by slow cooling.

580 The sequences of the oligonucleotide probes were synthesized by Eurofins Genomics and are
581 as following: 5'-ttaggttcacgtgtcgacatgaaaggagct-3', 5'-catatccatgtgtcgaaaacccgattttt-3' and 5
582 ' -catatccatgtgtcacaacccgggtttt-3. The binding of the purified proteins (≈ 150 ng) to the
583 Biotin-TEG labelled probes (20 fmol) was carried out using the LightShift Chemiluminescent
584 EMSA Kit (Thermo Scientific, Waltham, USA) in 20 µL reaction mixture containing 1X binding
585 buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5), 2.5% glycerol, 5 mM MgCl₂, 2 µg of poly
586 (dI-dC) and 0.05% NP-40. After incubation at 24°C for 30 min, the protein-probe mixture was
587 separated in a 4% polyacrylamide native gel at 100 V for 50 min then transferred to a Biodyne
588 B Nylon membrane (Thermo Scientific) by capillary action in 20X SSC buffer overnight. After
589 ultraviolet crosslinking (254 nm) for 90 s at 120 mJ.cm⁻². The migration of Biotin-TEG labelled
590 probes was detected using horseradish peroxidase-conjugated streptavidin in the LightShift
591 Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer's protocol, and
592 then exposed to X-ray film.

593
594 Phospholipid Extraction

595 Lipids were extracted from 18-days-old *Arabidopsis thaliana* shoots (Col-0) grown in the
596 presence or absence of Zn, following the Folch's method (Folch et al., 1957). The total
597 phosphorus (P) contained in lipids was measured using a spectrophotometer with an
598 absorbance at 830 nm. Lipid separation and quantification was performed using Thin Layer
599 Chromatography (TLC). The lipid composition was detected and quantified using a GAMAG
600 TLC SCANNER 3 (Muttenz, Switzerland), operating in the reflectance mode. The plates were
601 scanned at 715 nm after dipping in a solution of Blue Spray (Sigma, France) and heating for 3
602 min at 55 °C. The WinCat software program was used to scan bands, the different classes of
603 phospholipids (Fouret et al., 2015) were identified by comparing their retention factor (Rf) to
604 authentic standards and the quantities of each phospholipid were evaluated against the
605 corresponding calibration curve (Fouret et al., 2015).

606

607 In planta transactivation assay.

608 The *in planta* transactivation assay was performed in *N. benthamiana*. LPCAT Col-0 promoter
609 and LPCAT Col-0 mutated promoter were cloned pLPCAT1Col-0-For 5'-
610 ggggacaagtttgtacaaaaaagcaggcttcggtgtcgaaaacccgtttt-3'; pLPCAT1Col-0-Rev 5'-
611 ggggaccactttgtacaagaaagctgggtctgatcagagttacaacaggagag -3' ;pLPCAT1Col-0-mutated-For
612 5'-ggggacaagtttgtacaaaaaagcaggcttcggtgtcacaaacccgtttt-3'. The LPCAT1 promoters were
613 then fused to the β-GUS-encoding reporter gene using the Gateway system. These following
614 primers were used to clone the promoter of the zinc transporter ZIP4-For 5'-
615 ggggacaagtttgtacaaaaaagcaggcttcttggaaagtgaagtggattg-3'; ZIP4-REV 5'-
616 ggggaccactttgtacaagaaagctgggtctgatcatcgacgaagaccatgggaacaagagt -3' (Lin et al., 2016).
617 Each of these primer was then fused to β-GUS-encoding reporter gene. bZIP23 coding
618 sequences CDS was placed under the CaMV. 35S promoter. The 35S::C-YFP construct was
619 provided by Dr. Seung Y. Rhee (Bossi et al., 2017). Each construct was transformed into
620 Agrobacterium. Positive clones per construct were grown overnight at 28 °C, and then washed
621 four times in infiltration buffer (10 mM MgCl₂, 10 mM MES (pH 5.6) and 100 uM
acetosyringone). The effector construct and reporter construct (OD600 of 0.8), were co-infiltrated at a ratio of 9 to 1 in fully expanded leaves (3rd or 4th leaves) of five to six week-old tobacco plants. Each plasmid combination was infiltrated in one leaf from different plants. We used C-YFP as negative controls. Three independent infiltrations per combination resulting in 6 samples per construct. Three days after infiltration, leaves were collected, and the infiltrated areas in each leaf were excised and pooled into one sample. The GUS extraction and the GUS enzymatic activity measurements was performed as described by Bisso et al., (2017). The relative GUS enzymatic activity was determined by comparing the effect of bZIP23 TF and C-YFP protein on each promoter.

631

## 632 Statistical analysis

Statistical analysis of quantitative data was performed using the GraphPad prism 5.01 software program for Windows (GraphPad 156 Software, CA, USA, http://www.graphpad.com). For all the t-test analyses the difference was considered statistically significant when the test yielded a P-value < 0.05.

637

## 638 Acknowledgment

The authors are grateful to Dr. Santosh B. Satbhai and Bonnie Wohlrab for initial seed preparation of accessions and to Christian Goeschl for help with the Manhattan plots, to Drs Jérôme Lecomte and Christine Feillet-Coudray for their help with the lipid quantification. Thanks to Prof Pierre Berthomieu, Drs Patrick Doumas, Saber Kouas and Zaigham Shahzad for helpful discussions. Thanks to Dr Seung Y. Rhee LAB member (Carnegie Institution for Science, Stanford, USA) for help with the *in planta* transactivation assay. This work was funded by the Institut National de la Recherche Agronomique (INRA) and by the Région Languedoc-Roussillon: Chercheur d'Avenir 2015, Projet cofinancé par le Fonds Européen de Développement Régional to HR, the Austrian Academy of Sciences through the Gregor Mendel Institute and the Salk Institute for Biological Studies to WB, the Netherlands Genome

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649 Initiative ZonMW Horizon program Zenith project no. 40-41009-98-11084 supporting MA and

650 RA, and by an Iraq government doctoral fellowship for MK.

651 Competing interests

652 The authors declare that no competing interests exist.

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893
894 **Figure Legends**

895 **Figure 1. Genome-wide association (GWA) analysis of Arabidopsis shoot Pi concentration.** 223 Arabidopsis thaliana accessions were grown suplemented with zinc (+Zn) 896 or without zinc (-Zn) for 18 days under long day conditions, upon which shoot inorganic 897 phosphate (Pi) concentrations were determined. (A) Mean shoot Pi concentration of 898 Arabidopsis accessions in +Zn is plotted against the mean shoot Pi concentration of 899 Arabidopsis accessions in -Zn. (B, C) Manhattan plots of GWA analysis of Arabidopsis shoot 900 Pi concentration in -Zn (B) and +Zn (C). The five Arabidopsis chromosomes are indicated in 901 different colours. Each dot represents the -log10(P) association score of one single nucleotide 902 polymorphism (SNP). The dashed red line denotes an approximate false discovery rate 10% 903 threshold. Boxes indicate the location of the *LPCAT1* (red) quantitative trait loci (QTL). (D) 904 Gene models (upper panel) and SNP -log10(P) scores (lower panel) in the genomic region 905 surrounding the GWA QTL at the *LPCAT1* (E); 5' and 3' indicate the different genomic DNA 906 strands and orientation of the respective gene models. (E) Distribution of Pi concentrations in 907 accessions with the phenotype A versus accessions with phenotype G. Asterisk indicates a 908 significant difference between the two groups of accessions of P < 0.01. 909

910

911 **Figure 2. Loss of function mutation of Lyso-PhosphatidylCholine AcylTransferase 1 (LPCAT1), and not At1g12650, affects shoot Pi concentration in a Zn supply and bZIP33 dependent manner.** (A) Shoot Pi concentration of 18-days-old Col-0 wild-type plants, lpcat1 912 and At1g12650 mutants grown in +Zn or -Zn conditions. (B) Gene structure of *LPCAT1*. The 913 grey box represents the promoter region, green boxes are 5' and 3' untranslated regions, 914 black boxes represent exons, and black lines represent introns, the arrow head indicates the 915 direction of transcription, ATG indicates the start codon. The Zinc Deficiency Response 916 Element (ZDRE) binding site for bZIP19 and bZIP23, and the newly identified biding site for 917 bZIP23 are indicated. (C) Differential binding of bZIP19 and bZIP23 to two promoter regions of 918 LPCAT1 gene. EMSA analysis on 30-bp promoter fragments from motif present in *LPCAT1* 919

920
promoter of contrasting accessions showed in (A). (D) Relative *LPCAT1* transcript abundance (-Zn/+Zn) in Col-0 wild-type plants grown on +Zn or -Zn agar medium for 6, 12 and 18 days. (E) Relative *LPCAT1* transcript abundance in Col-0 wild-type plants, *bzip19*, *bzip23*, and *bzip19/bzip23* double mutants grown on +Zn or -Zn agar medium for 18 days. The relative mRNA levels was quantified by RT-qPCR and normalized to the *Ubiquitin10* reference mRNA level (*UBQ10*: At4g05320). (F) Shoot Pi concentration in Col-0 wild-type plants, *bzip19* and *bzip19/bzip23* double mutants grown on +Zn or -Zn agar medium for 18 days. Values are means of three to six biological replicates. Individual measurements were obtained from the analysis of shoots collected from a pool of 10 plants. Error bars indicate SD; One and two asterisks indicate a significant difference with WT plants (ANOVA and Tukey test) of P < 0.05 and P < 0.01, respectively.

**Figure 3. Identification of a new binding motif specific for bZIP23, and the variation of LPCAT1 gene expression between genotypes in -Zn condition.** (A) Sequence comparison for ZDRE and the new binding site motif for bZIP23 in the promoter of accession with high ratio of Pi accumulation in -Zn/+Zn (Col-0, Ang-0, CICB-5, Est-1, RRS-10) and low Pi accumulation ratio - Zn/+Zn (Sap-0, Ts-1, Br-0 and PHW-2). (B) Differential binding of bZIP19 and bZIP23 to a specific ZDRE motif of *LPCAT1* (GTGTCACA). (C-D) *in planta* transactivation assay. (C) 35S:bZIP23 and 35S::C-YFP were used as effectors. pLPCAT1: the native Col-0 LPACT1 promoter (with « GTGTCGAA » as new ZDRE), mpLPCAT1: a modified (point mutation) version of the Col-0 promoter to only contain the new ZDRE of Sap-0 (« GTGTCACA ») ; pZIP4 ; promoter of the zinc transporter ZIP4 gene. Each pLPCAT1 (native, Col-0), mpLPCAT1 (mutated version) or pZIP4 promoter was fused to a β-glucuronidase (GUS)-encoding reporter gene (reporter). (D) The effect of bZIP23 TF on the activity of each promoter pLPCAT1, mpLPCAT1 or pZIP4 was determined by measuring GUS activity. The effect of C-YFP protein on the activity of each promoter pLPCAT1, mpLPCAT1 or pZIP4 was used as a control to determine the basal level of GUS activity for each promoter. Comparing the effect of bZIP23 TF and C-YFP protein on each promoter enabled the determination of the
949 relative GUS activity. Error bars represent standard error from 3 independent experiments.
950 The asterisks indicate that the relative GUS activity is statistically different from the YFP
951 control (p-value <0.01, t-test). (E) Relative *bZIP23* and *LPCAT1* transcripts abundance in -Zn
952 and +Zn conditions. Col-0, Ang-0, CICB-5, Est-1, RRS-10, Sap-0, Ts-1, Br-0 and PHW-2
953 genotypes were grown on +Zn or -Zn agar medium. The relative mRNA level was quantified by
954 RT-qPCR and normalized to the *Ubiquitin10* reference mRNA level (UBQ10: At4g05320).
955 Values are means of six biological replicates. Individual measurements were obtained from the
956 analysis of shoots collected from a pool of 20 plants. Error bars indicate SD; one and two
957 asterisk indicates a significant difference with Col-0 plants (ANOVA and Tukey test) of P <
958 0.05 and P < 0.01 respectively.

959
960 **Figure 4. Natural allelic variation of LPCAT1 locus causes phenotypic variation of Pi**
961
962 accumulation in Zn deficiency conditions. (A) Schematic representation of the transgenic
963 constructs used to complement the *lpcat1* null mutant (Col-0 background). (B) Shoot Pi
964 concentration (–Zn / +Zn) of 18-days-old Col-0 wild-type plants, *lpcat1* mutant transformed
965 with empty vector, or with constructs schematized in (A) grown in +Zn or –Zn conditions. (C)
966 The ANOVA results are presented in the table. Significative codes: ‘***’ 0.001 and ‘*’ 0.05.
967 Relative LPCAT1 transcript abundance in wild-type plants (Col-0 background) and the
968 transgenic lines generated using the construct schematized in (A) grown on +Zn or -Zn agar
969 medium. The relative mRNA levels was quantified by RT-qPCR and normalized to the
970 *Ubiquitin10* reference mRNA level (UBQ10: At4g05320). Values are means of three to
971 biological replicates. Individual measurements were obtained from the analysis of shoots
972 collected from a pool of six plants. Error bars indicate SD; asterisks indicates a significant
difference with Col-0 plants (ANOVA and Tukey test) of P < 0.05.

973
974 **Figure 5. Loss of function mutations of LPCAT1 affect the lysoPC/PC ratio in –Zn**
975 conditions. (A) Schematic representation of the biochemical function of LPCAT1, which
976 catalyses the formation of phosphatidylcholine (PC) from lyso-PC and long-chain acyl-CoA.
977 (B) Lyso-PC concentration (C) PC concentration (D) Lyso-PC/PC concentration ratios of Col-0
wild-type plants, *bzip23* and *lpcat1* mutant lines grown in +Zn or -Zn conditions for 18 days.

Individual measurements were obtained from the analysis of shoots collected from a pool of five plants. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.05) between mutants and Col-0 are indicated with asterisks.

983

**Figure 6. Effect of the polymorphisms in the regulatory region of LPCAT1 on the change in LPC/PC ratios in -Zn conditions.** (A) Lyso-PC concentration (B) PC concentration (C) Lyso-PC/PC concentration ratios of Sap-0, Col-0 wild-type plants, and *lpcat1* expressing pLPCAT1<sup>Col-0</sup>::LPCAT1<sup>Col-0</sup>, pLPCAT1<sup>Col-0</sup>::LPCAT1<sup>Sap-0</sup>, pLPCAT1<sup>Sap-0</sup>::LPCAT1<sup>Col-0</sup>, pLPCAT1<sup>Sap-0</sup>::LPCAT1<sup>Sap-0</sup> constructs and *lpcat1* transformed with empty lines grown in +Zn or -Zn conditions for 18 days. Individual measurements were obtained from the analysis of shoots collected from a pool of five plants. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.05) between mutants and Col-0 are indicated with asterisks.

993

**Figure 7. Loss of function mutations of LPCAT1 show enhanced expression of PHT1;1 when compared to Col-0 wild-type plants.** (A, B) Genome-wide association (GWA) analysis of Arabidopsis shoot Pi concentration. 223 Arabidopsis thaliana accessions were grown on agar medium supplemented with zinc (+Zn) or without zinc (-Zn) for 18 days under long day conditions, upon which shoot inorganic phosphate (Pi) concentrations were determined. Manhattan plots of GWA analysis of Arabidopsis shoot Pi concentration in -Zn (A) and +Zn (B). The five Arabidopsis chromosomes are indicated in different colours. Each dot represents the -log10(P) association score of one single nucleotide polymorphism (SNP). The dashed red line denotes an approximate false discovery rate 10% threshold. Boxes indicate the location of the PHT1 (blue) quantitative trait loci (QTL). (C) Gene models (upper panel) and SNP - log10(P) scores (lower panel) in the genomic region surrounding the GWA QTL at the or PHT1 locus; 5' and 3' indicate the different genomic DNA strands and orientation of the
1006 respective gene models. (D) Relative expression level of all members of the Arabidopsis *PHT1* gene family in shoots of 18-days-old Col-0 wild-type plants and *lpcat1* mutants grown on -Zn agar medium compared to their expression on +Zn. mRNA accumulation was quantified by RT-qPCR, normalized to the mRNA level of the *UBIQUITIN10* reference gene (*UBQ10*: At4g05320) and expressed as relative values against its *UBQ10* normalized mRNA level of Col-0 grown in +Zn medium (control). (E) Shoot Pi concentration of 18-days-old Col-0 wild-type plants, *pht1;1*, *pht1;2*, and *pht1;3* mutants grown in +Zn or -Zn conditions. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.05 and P < 0.01) are indicated by one or two asterisks.

1015

1016 Supplementary Figure

1017 **Figure 1-figure supplement 1. mRNA abundance of Zn-responsive genes ZIP4 and ZIP12**
1018 **in roots of Col-0 plants grown in presence and absence of Zn.** Reverse transcriptase
1019 qPCR analyses of transcript levels changes in response to Zn-deficiency of the genes ZIP4
1020 (At1g10970) and ZIP12 (At5g62160) in shoots of Arabidopsis (Col-0). Seedlings were grown
1021 on vertical agar plate in presence or absence of Zn for 18 days. Transcript levels of these
1022 genes are expressed relative to the average transcript abundance of the UBIQUITIN10
1023 (UBQ10; At4g05320) that was used as an internal control. Every data point was obtained from
1024 the analysis of shoots collected from a pool of six plants. Data presented are means of three
1025 biological replicates ± SE. Asterisks indicate statistically significant differences compared to
1026 the +Zn condition for each gene analyzed (P <0.05).

1027

1028 **Figure 1-figure supplement 2. Genome-wide association (GWA) analysis of Arabidopsis**
1029 **shoot Pi concentration.** 223 Arabidopsis thaliana accessions were grown supplemented with
1030 zinc (+Zn) or without zinc (-Zn) for 18 days under long day conditions, upon which shoot
1031 inorganic phosphate (Pi) concentrations were determined. (A, B) Histogram of the frequency
1032 distribution of mean shoot Pi concentration of Arabidopsis accessions in +Zn (A) and -Zn (B).
1033 (C, D) Manhattan plots of GWA analysis of Arabidopsis shoot Pi concentration in -Zn (C) and
1034 +Zn (D) generated using 1001 Genomes imputed SNPs. Each dot represents the -log10(P) association score of one single nucleotide polymorphism (SNP). Boxes indicate the location of the *LPCAT1* (red) quantitative trait loci (QTL).

1037

1038 **Figure 1-figure supplement 3. Haplotype analysis of region around SNP C1P4306845.** (upper panel) Clusterplot represents the haplotype blocks according to fastphase2 for SNPs 1039 25 kb up and downstream of the significantly associated SNP. Colours represent a specific 1040 haplotype. X-axis: Accessions, Y-axis: SNPs. The horizontal blue line indicates the position of 1041 the significant SNP. The red box indicates the LPCAT gene model. The blue arrow represents 1042 the transcription direction of the gene model (LPCAT is on the - strand). (lower panel) 1043 Normalized Pi content in -Zn condition of each accession (using the R scale function). Colour 1044 indicates the genotype of the accession at SNP C1P4306845 (top GWAS hit), whereby red 1045 colour indicates the minor SNP allele that is associated with high Pi content in -Zn. 1046

1047

1048 **Figure 3-figure supplement 1. Shoot Pi concentrations in Arabidopsis accessions 1049 grouped by new ZDRE motif.** Shoot Pi concentrations of *Arabidopsis thaliana* accessions 1050 grown on agar medium supplemented with zinc (+Zn) (A) or without zinc (-Zn) (B) for 18 days 1051 under long day conditions. Data same as in Supplementary file 3. Grouping of accessions is 1052 based on their ZDRE binding motif allele TGTC**AAA**, TGTC**ACA** and TGTC**GAA** respectively. 1053 Asterisk indicates a significant difference according to Student's T-test P < 0.01.

1054

1055 **Figure 7-figure supplement 1. Relative expression level of all members of the 1056 Arabidopsis *PHT1* gene family.** *Arabidopsis thaliana* Col-0, *lpcat1-1* and *lpcat1-2* mutant 1057 lines were grown on agar medium supplemented with zinc (+Zn) for 18 days under long day 1058 conditions. mRNA level was quantified by RT-qPCR and normalized to the *Ubiquitin10* 1059 reference mRNA level (*UBQ10*: At4g05320) and expressed as relative values against the 1060 *UBQ10* normalized mRNA level in Col-0. Values are means of six biological replicates. 1061 Individual measurements were obtained from the analysis of shoots collected from a pool of 20
1062 plants. Error bars indicate SD; one and two asterisk indicates a significant difference with Col-
1063 0 plants (ANOVA and Tukey test) of P < 0.05 and P < 0.01 respectively.

1064
1065 **Figure 7-figure supplement 2. High Affinity of Phosphate Transporter (PHT1;1) gene expression analysis.** Loss of function mutations of *LPCAT1* show enhanced expression of *PHT1;1* when compared to Col-0 wild-type plants. Relative expression level of *PHT1;1* gene in roots of 18-days-old Col-0 wild-type plants and *lpcat1* mutants grown on -Zn agar medium. mRNA accumulation was quantified by RT-qPCR, normalized to the mRNA level of the *UBIQUITIN10* reference gene (*UBQ10*: At4g05320) and expressed as relative values against Col-0 grown in +Zn medium (control). Individual measurements were obtained from the analysis of roots collected from a pool of five plants. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.01) are indicated by two asterisks.
1066
1067
1068
1069
1070
1071
1072
1073
1074

1075
1076 Supplement Table

1077 **Supplement file 1.** Shoots Pi concentration in the 223 *Arabidopsis thaliana* accessions grown
1078 in presence or absence of zinc.

1079 **Supplement file 2.** Coordinates for the significant SNPs associated with Pi concentration in
1080 shoots in Zn conditions.

1081 **Supplement file 3.** List of new ZDRE motif in the *Arabidopsis thaliana* accessions and the
1082 shoot Pi content in presence (+Zn) or absence of Zn (-Zn).
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>

**Figure 1. Genome-wide association (GWA) analysis of Arabidopsis shoot Pi concentration.** 223 *Arabidopsis thaliana* accessions were grown suplemented with zinc (+Zn) or without zinc (-Zn) for 18 days under long day conditions, upon which shoot inorganic phosphate (Pi) concentrations were determined. (A) Mean shoot Pi concentration of Arabidopsis accessions in +Zn is plotted against the mean shoot Pi concentration of Arabidopsis accessions in -Zn. (B, C) Manhattan plots of GWA analysis of Arabidopsis shoot Pi concentration in -Zn (B) and +Zn (C). The five Arabidopsis chromosomes are indicated in different colours. Each dot represents the -log10(P) association score of one single nucleotide polymorphism (SNP). The dashed red line denotes an approximate false discovery rate 10% threshold. Boxes indicate the location of the LPCAT1 (red) quantitative trait loci (QTL). (D) Gene models (upper panel) and SNP -log10(P) scores (lower panel) in the genomic region surrounding the GWA QTL at the LPCAT1 (E); 5' and 3' indicate the different genomic DNA strands and orientation of the respective gene models. (E) Distribution of Pi concentrations in accessions with the phenotype A versus accessions with phenotype G. Asterisk indicates a significant difference between the two groups of accessions of P < 0.01.
<figure><img src="image_3.png" /><figcaption>Figure 1-figure supplement 1</figcaption></figure>

**Figure 1-figure supplement 1. mRNA abundance of Zn-responsive genes ZIP4 and ZIP12 in roots of Col-0 plants grown in presence and absence of Zn.** Reverse transcriptase qPCR analyses of transcript levels changes in response to Zn-deficiency of the genes *ZIP4* (At1g10970) and *ZIP12* (At5g62160) in shoots of Arabidopsis (Col-0). Seedlings were grown on vertical agar plate in presence or absence of Zn for 18 days. Transcript levels of these genes are expressed relative to the average transcript abundance of the *UBIQUITIN10* (UBQ10; At4g05320) that was used as an internal control. Every data point was obtained from the analysis of shoots collected from a pool of six plants. Data presented are means of three biological replicates ± SE. Asterisks indicate statistically significant differences compared to the +Zn condition for each gene analyzed (P <0.01).
<figure><img src="image_4.png" /><figcaption>**Figure 1-figure supplement 2. Genome-wide association (GWA) analysis of Arabidopsis shoot Pi concentration.** 223 Arabidopsis thaliana accessions were grown supplemented with zinc (+Zn) or without zinc (-Zn) for 18 days under long day conditions, upon which shoot inorganic phosphate (Pi) concentrations were determined. (A, B) Histogram of the frequency distribution of mean shoot Pi concentration of Arabidopsis accessions in +Zn (A) and -Zn (B). (C, D) Manhattan plots of GWA analysis of Arabidopsis shoot Pi concentration in -Zn (C) and +Zn (D) generated using 1001 Genomes imputed SNPs. Each dot represents the -log10(P) association score of one single nucleotide polymorphism (SNP). Boxes indicate the location of the LPCAT1 (red) quantitative trait loci (QTL).</figcaption></figure>
Figure 1-figure supplement 3

<figure><img src="image_5.png" /><figcaption>**Figure 1-figure supplement 3. Haplotype analysis of region around SNP C1P4306845.** (upper panel) Clusterplot represents the haplotype blocks according to fastphase2 for SNPs 25 kb up and downstream of the significantly associated SNP. Colours represent a specific haplotype. X-axis: Accessions, Y-axis: SNPs. The horizontal blue line indicates the position of the significant SNP. The red box indicates the LPCAT gene model. The blue arrow represents the transcription direction of the gene model (LPCAT is on the - strand). (lower panel) Normalized Pi content in -Zn condition of each accession (using the R scale function). Colour indicates the genotype of the accession at SNP C1P4306845 (top GWAS hit), whereby red colour indicates the minor SNP allele that is associated with high Pi content in -Zn.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>

**Figure 2.** Loss of function mutation of *Lyso-PhosphatidylCholine AcylTransferase 1 (LPCAT1)*, and not *At1g12650*, affects shoot Pi concentration in a Zn supply and bZIP23 dependent manner. (A) Shoot Pi concentration of 18-days-old Col-0 wild-type plants, *lpcat1* and *At1g12650* mutants grown in +Zn or -Zn conditions. (B) Gene structure of *LPCAT1*. The grey box represents the promoter region, green boxes are 5' and 3' untranslated regions, black boxes represent exons, and black lines represent introns, the arrow head indicates the direction of transcription, ATG indicates the start codon. The Zinc Deficiency Response Element (ZDRE) binding site for *bZIP19* and *bZIP23*, and the newly identified biding site for *bZIP23* are indicated. (C) Differential binding of *bZIP19* and *bZIP23* to ZDRE motif and *bZIP23* specific motif (GTGTCGAA) of *LPCAT1*. (D) Relative *LPCAT1* transcript abundance (-Zn/+Zn) in Col-0 wild-type plants grown on +Zn or -Zn agar medium for 6, 12 and 18 days. (E) Relative *LPCAT1* transcript abundance in Col-0 wild-type plants, *bzip19*, *bzip23*, and *bzip19 bzip23* double mutants grown on +Zn or -Zn agar medium for 18 days. The relative mRNA levels was quantified by RT-qPCR and normalized to the *Ubiquitin10* reference mRNA level (UBQ10: At4g05320). (F) Shoot Pi concentration in Col-0 wild-type plants, *bzip19* and *bzip19/bzip23* double mutants grown on +Zn or -Zn agar medium for 18 days. Values are means of six biological replicates. Individual measurements were obtained from the analysis of shoots collected from a pool of 10 plants. Error bars indicate SD; One and two asterisks indicates a significant difference with WT plants (ANOVA and Tukey test) of *P* < 0.05 and *P* < 0.01, respectively.

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>Figure 3</figcaption></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

**Figure 3.** Identification of a new binding motif specific for bZIP23, and the variation of LPCAT1 gene expression between genotypes in -Zn condition. (A) Sequence comparison for ZDRE and the new binding site motif for bZIP23 in the promoter of accession with high ratio of Pi accumulation in -Zn/+Zn (Col-0, Ang-0, CICB-5, Est-1, RRS-10) and low Pi accumulation ratio - Zn/+Zn (Sap-0, Ts-1, Br-0 and PHW-2). (B) Differential binding of bZIP19 and bZIP23 to a specific ZDRE motif of LPCAT1 (GTGTCACA). (C-D) in planta transactivation assay. (C) 35S:bZIP23 and 35S::C-YFP were used as effectors. pLPCAT1: the native Col-0 LPACT1 promoter (with « GTGTCGAA » as new ZDRE), mpLPCAT1: a modified (point mutation) version of the Col-0 promoter to only contain the new ZDRE of Sap-0 (« GTGTCACA »); pZIP4; promoter of the zinc transporter ZIP4 gene. Each pLPCAT1 (native, Col-0), mpLPCAT1 (mutated version) or pZIP4 promoter was fused to a β-glucuronidase (GUS)-encoding reporter gene (reporter). (D) The effect of bZIP23 TF on the activity of each promoter pLPCAT1, mpLPCAT1 or pZIP4 was determined by measuring GUS activity. The effect of C-YFP protein on the activity of each promoter pLPCAT1, mpLPCAT1 or pZIP4 was used as a control to determine the basal level of GUS activity for each promoter. Comparing the effect of bZIP23 TF and C-YFP protein on each promoter enabled the determination of the relative GUS activity. Error bars represent standard error from 3 independent experiments. The asterisks indicate that the relative GUS activity is statistically different from the YFP control (p-value <0.01, t-test). (E) Relative bZIP23 and LPCAT1 transcripts abundance in -Zn and +Zn conditions. Col-0, Ang-0, CICB-5, Est-1, RRS-10, Sap-0, Ts-1, Br-0 and PHW-2 genotypes were grown on +Zn or -Zn agar medium. The relative mRNA level was quantified by RT-qPCR and normalized to the *Ubiquitin10* reference mRNA level (UBQ10: At4g05320). Values are means of six biological replicates. Individual measurements were obtained from the analysis of shoots collected from a pool of 20 plants. Error bars indicate SD; one and two asterisk indicates a significant difference with Col-0 plants (ANOVA and Tukey test) of P < 0.05 and P < 0.01 respectively.
<figure><img src="image_11.png" /><figcaption>**Figure 3- figure supplement 1. Shoot Pi concentrations in Arabidopsis accessions grouped by new ZDRE motif.** Shoot Pi concentrations of Arabidopsis thaliana accessions grown on agar medium supplemented with zinc (+Zn) (A) or without zinc (-Zn) (B) for 18 days under long day conditions. Data same as in Table S3. Grouping of accessions is based on their ZDRE binding motif allele TGTCAAA, TGTCACA and TGTCGAA respectively. Asterisk indicates a significant difference according to Student's T-test P < 0.01.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Figure 4</figcaption></figure>

<figure><img src="image_13.png" /></figure>

**Figure 4.** Natural allelic variation of LPCAT1 locus causes phenotypic variation of Pi accumulation in Zn deficiency conditions. (A) Schematic representation of the transgenic constructs used to complement the *lpcat1* null mutant (Col-0 background). (B) Shoot Pi concentration ratios (–Zn / +Zn) of 18-days-old Col-0 wild-type plants, *lpcat1* mutant transformed with empty vector, or with constructs schematized in (A) grown in +Zn or –Zn conditions. Two-way ANOVA was used to test the impact of Promoter (Col-0 v Sap-0), ORF(LPCAT1-Col-0 v LPCAT1-Sap-0) and their interaction on Pi aconcentration. The ANOVA results are presented in the table. Significative codes: '*****' 0.001 and '*' 0.05. (C) Relative *LPCAT1* transcript abundance in wild-type plants (Col-0 background) and the transgenic lines generated using the construct schematized in (A) grown on +Zn or -Zn agar medium. The relative mRNA levels was quantified by RT-qPCR and normalized to the *Ubiquitin10* reference mRNA level (*UBQ10*: At4g05320). Values are means of six biological replicates. Individual measurements were obtained from the analysis of shoots collected from a pool of six plants. Error bars indicate SD; letters indicates a significant difference between empty vector, Col-0 and trasngenitic lines (ANOVA and Tukey test) of *P* < 0.05.
<figure><img src="image_14.png" /><figcaption>Figure 5</figcaption></figure>

**Figure 5.** Loss of function mutations of LPCAT1 affect the lysoPC-PC ratio in -Zn conditions.

(A) Schematic representation of the biochemical function of *LPCAT1*, which catalyses the formation of phosphatidylcholine (PC) from lyso-PC and long-chain acyl-CoA. (B) Lyso-PC concentration (C) PC concentration (D) Lyso-PC/PC concentration ratios of Col-0 wild-type plants, *bzip23* and *lpcat1* mutant lines grown in +Zn or -Zn conditions for 18 days. Individual measurements were obtained from the analysis of shoots collected from a pool of five plants. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.05). between mutants and Col-0 are indicated with asterisks.
<figure><img src="image_15.png" /><figcaption>Figure 6</figcaption></figure>

**Figure 6.** Effect of the polymorphisms in the regulatory region of LPCAT1 on the change in LPC/PC ratios in –Zn conditions. (A) Lyso-PC concentration (B) PC concentration (C) Lyso-PC/PC concentration ratios of Sap-0, Col-0 wild-type plants, and *lpcat1* expressing pLPCAT1Col-0::LPCAT1Col-0, pLPCAT1Col-0::LPCAT1Sap-0, pLPCAT1Sap-0::LPCAT1Col-0, pLPCAT1Sap-0::LPCAT1Sap-0 constructs and *lpcat1* transformed with empty lines grown in +Zn or -Zn conditions for 18 days. Individual measurements were obtained from the analysis of shoots collected from a pool of five plants. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.05) between mutants and Col-0 are indicated with asterisks.
<figure><img src="image_16.png" /><figcaption>Figure 7. Loss of function mutations of *LPCAT1* show enhanced expression of *PHT1;1* when compared to Col-0 wild-type plants. (A, B) Genome-wide association (GWA) analysis of Arabidopsis shoot Pi concentration. 223 Arabidopsis thaliana accessions were grown agar medium supplemented with zinc (+Zn) or without zinc (-Zn) for 18 days under long day conditions, upon which shoot inorganic phosphate (Pi) concentrations were determined. Manhattan plots of GWA analysis of Arabidopsis shoot Pi concentration in -Zn (A) and +Zn (B). The five Arabidopsis chromosomes are indicated in different colours. Each dot represents the -log10(P) association score of one single nucleotide polymorphism (SNP). The dashed red line denotes an approximate false discovery rate 10% threshold. Boxes indicate the location of the *PHT1* (blue) quantitative trait loci (QTL). (C) Gene models (upper panel) and SNP -log10(P) scores (lower panel) in the genomic region surrounding the GWA QTL at the or *PHT1* locus; 5' and 3' indicate the different genomic DNA strands and orientation of the respective gene models. (D) Relative expression level of all members of the Arabidopsis *PHT1* gene family in shoots of 18-days-old Col-0 wild-type plants and *lpcat1* mutants grown on -Zn agar medium compared to their expression on +Zn. mRNA accumulation was quantified by RT-qPCR, normalized to the mRNA level of the *UBIQUITIN10* reference gene (UBQ10: At4g05320) and expressed as relative values against its UBQ10 normalized mRNA level of Col-0 grown in +Zn medium (control). (E) Shoot Pi concentration of 18-days-old Col-0 wild-type plants, *pht1;1*, *pht1;2*, and *pht1;3* mutants grown in +Zn or -Zn conditions. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.05 and P < 0.01) are indicated by one or two asterisks.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Figure 7-figure supplement 1</figcaption></figure>

**Figure 7-figure supplement 1. Relative expression level of all members of the Arabidopsis *PHT1* gene family.** *Arabidopsis thaliana* Col-0, *lpcat1-1* and *lpcat1-2* mutant lines were grown agar medium supplemented with zinc (+Zn) for 18 days under long day conditions. mRNA level was quantified by RT-qPCR and normalized to the *Ubiquitin10* reference mRNA level (*UBQ10*: At4g05320) and expressed as relative values against the *UBQ10* normalized mRNA level in Col-0. Values are means of six biological replicates. Individual measurements were obtained from the analysis of shoots collected from a pool of 20 plants. Error bars indicate SD; one and two asterisk indicates a significant difference with Col-0 plants (ANOVA and Tukey test) of P < 0.05 and P < 0.01 respectively.
<figure><img src="image_18.png" /><figcaption>Figure 7-figure supplement 2</figcaption></figure>

**Figure 7-figure supplement 2. High Affinity of Phosphate Transporter (PHT1;1) gene expression analysis.**

Loss of function mutations of LPCAT1 show enhanced expression of *PHT1;1* when compared to Col-0 wild-type plants. Relative expression level of *PHT1;1* gene in roots of 18-days-old Col-0 wild-type plants and *lpcat1* mutants grown on -Zn agar medium. mRNA accumulation was quantified by RT-qPCR, normalized to the mRNA level of the *UBIQUITIN10* reference gene (*UBQ10*: At4g05320) and expressed as relative values against Col-0 grown in +Zn medium (control). Individual measurements were obtained from the analysis of roots collected from a pool of five plants. Data are mean ± SD of three biological replicates. Statistically significant differences (ANOVA and Tukey test, P < 0.01) are indicated by two asterisks.