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# The vacuolar sulfate transporter PsSULTR4 is a key determinant of seed yield and protein composition in pea

Fanélie Bachelet, Myriam Sanchez, Delphine Aimé, Florence Naudé, Nadia Rossin, Alain Ourry, Chrystel Deulvot, Christine Le Signor, Vanessa Vernoud, Fabrice Neiers, et al.

► To cite this version:

Fanélie Bachelet, Myriam Sanchez, Delphine Aimé, Florence Naudé, Nadia Rossin, et al. The vacuolar sulfate transporter PsSULTR4 is a key determinant of seed yield and protein composition in pea. The Plant Journal, 2024, 119 (6), pp.2919-2936. 10.1111/tpj.16961. hal-04676560

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https://hal.inrae.fr/hal-04676560v1

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Distributed under a Creative Commons Attribution 4.0 International License
The Plant Journal (2024) 119, 2919–2936

doi: 10.1111/tpj.16961

# The vacuolar sulfate transporter PsSULTR4 is a key determinant of seed yield and protein composition in pea

Fanélie Bachelet<fnref n="1" /> ID, Myriam Sanchez<fnref n="1" />, Delphine Aime<fnref n="1" />, Florence Naudé<fnref n="1" />, Nadia Rossin<fnref n="1" />, Alain Ourry<fnref n="2" /> ID, Chrystel Deulvot<fnref n="1" />,<fnref n="3" />, Christine Le Signor<fnref n="1" /> ID, Vanessa Vernoud<fnref n="1" />, Fabrice Neiers<fnref n="4" /> ID, Markus Wirtz<fnref n="5" />,* ID and Karine Gallardo-Guerrero<fnref n="1" />,* ID

Received 22 March 2024; revised 19 June 2024; accepted 23 July 2024; published online 8 August 2024.

## SUMMARY

Pea is a grain legume crop with a high potential to accelerate the food transition due to its high seed protein content and relatively well-balanced amino acid composition. The critical role of external sulfur (S) supply in determining seed yield and seed quality in pea makes it essential to understand the impact of whole plant S management on the trade-off between these two traits. Here, we investigated the physiological relevance of vacuolar sulfate remobilization by targeting PsSULTR4, the only pea sulfate transporter showing substantial similarity to the vacuolar sulfate exporter AtSULTR4;1. Five mutations in PsSULTR4 were identified by TILLING (Targeting Induced Local Lesions IN Genomes), two of which, a loss of function (W78*) and a missense (E568K), significantly decreased seed yield under S deprivation. We demonstrate that PsSULTR4 triggers S distribution from source tissues, especially lower leaves, to reproductive organs to maintain seed yield under S deficiency. Under sufficient S supply, sultr4 seeds display lower levels of the S-rich storage protein PA1 at maturity. They also overaccumulate sulfate in the endosperm at the onset of seed filling. These findings uncover a role of PsSULTR4 in the remobilization of vacuolar sulfate during embryo development, allowing the efficient synthesis of S-rich proteins. Our study uncovers that PsSULTR4 functions (i) in source tissues to remobilize stored vacuolar sulfate for seed production under low S availability and (ii) in developing seeds well supplied with S to fine-tune sulfate remobilization from the endosperm as a critical control point for storage activities in the embryo.

**Keywords:** Pisum sativum, sulfate transport, vacuolar efflux, legumes, seed yield, protein quality, sulfur-rich seed proteins.

## INTRODUCTION

In the last decades, lowered anthropogenic emissions of S dioxide caused a substantial decrease in soil sulfur (S) deposition. The resulting S deficiency of soils is a growing concern, as this macronutrient is essential for plant growth, productivity, and seed quality (Aulakh, 2003; Kopriva, 2004). The impact of lowered S-supply is well documented in pea (Pisum sativum L.) (Chandler et al., 1983, 1984; Henriet et al., 2019; Higgins et al., 1986), which is a grain legume crop with high potential for accelerating the transition to a

more sustainable and healthy food. Grain legumes are capable of accumulating high amounts of seed storage proteins (SSP, between 20 and 30% in pea, Daba & Morris, 2022) even in the absence of nitrogen (N) fertilization, thanks to their symbiosis with soil bacteria, rhizobia, which can fix atmospheric N (Beringer et al., 1979). In pea, the most abundant seed proteins (i.e. SSP) have been classified as 7S globulins (vicilins and convicilins), 11S globulins (legumins), and 2S albumins (PA1 and PA2) (Osborne, 1924). The S supply of the mother plant strongly influences SSP

<footnote n="1">¹Agroécologie, Institut National de l'Agriculture, de l'Alimentation et de l'Environnement (INRAE), Institut Agro, Université de Bourgogne, Université de Bourgogne Franche-Comté, Dijon 21000, France,</footnote>

<footnote n="2">²Normandie Université, UNICAEN INRAE, UMR 950 Ecophysiologie Végétale, Agronomie et Nutritions N, C, S, Caen, France,</footnote>

<footnote n="3">³Plateforme DlmaCell, Agroécologie, INRAE, Institut Agro, Université de Bourgogne, Université de Bourgogne Franche-Comté, Dijon 21000, France,</footnote>

<footnote n="4">⁴Centre des Sciences du Goût et de l'Alimentation (CSGA), Université de Bourgogne, INRAE, CNRS, Institut Agro, Dijon, France, and</footnote>

<footnote n="5">⁵Centre for Organismal Studies (COS), University of Heidelberg, Heidelberg 69120, Germany</footnote>

<footnote>*For correspondence (e-mail karine.gallardo-guerrero@inrae.fr and markus.wirtz@cos.uni-heidelberg.de).</footnote>
composition in seeds. Indeed, genes encoding the pea SSP with the highest content of S-containing amino acids (SAA) are down-regulated in response to S deficiency (Chandler et al., 1983, 1984; Henriet et al., 2019). The decrease of S-rich 2S albumins and 11S globulins in mature seeds is compensated by an increased accumulation of the S-poor 7S globulins (Chandler et al., 1983, 1984; Henriet et al., 2019). Because the least abundant amino acids of pea seeds are SAA (Met, essential amino acid; and Cys, semi-essential) (Hertzler et al., 2020), S deficiency will undoubtedly have a very negative impact on the amino acid balance of seeds, thus affecting their nutritional value. In contrast to nitrate, sulfate can be reduced in the seed embryo, which enables the embryo to *de novo* synthesize SAA for translation of SSP (Tabe & Droux, 2001). Understanding sulfate management by pea plants is therefore essential for optimizing S-use efficiency in this grain legume crop and would enable us to stabilize protein yield and the nutritional quality of pea seeds. The latter's relevance is reinforced by the emerging interest to replace part of animal proteins with plant proteins in the human diet while reducing fertilizer applications in the fields to decrease environmental pollution.

The S status of plant tissues, including seeds, depends on the availability of S in soils and efficient transport systems for S uptake and S distribution within and between cells (Buchner, 2004). S is taken up from the soil as sulfate, which can move through the xylem and phloem and between vessels and tissues. Exclusively in plastids, sulfate is reduced to sulfide, which is subsequently incorporated into Cys and Met (Wirtz & Droux, 2005). Upon sufficient S supply, sulfate is stored in the central vacuole of plant cells, allowing plants to store substantial amounts of sulfate to endure S-limiting conditions. Due to this sophisticated subcellular compartmentalization, sulfate homeostasis is tightly regulated by active transport systems (Li et al., 2020). There are 13 and 14 genes encoding putative sulfate transporters (SULTR from groups 1 to 5) in the genomes of rice (monocots) and Arabidopsis (eudicots, rosid clade), respectively (Kopriva et al., 2007). These transporters have been classified into five groups based on phylogenetic analyses (Hawkesford, 2003), and a role in sulfate transport has been established for SULTR of groups 1 to 4 (Takahashi, 2019; Takahashi et al., 2012). Briefly, SULTRs of groups 1-3 are involved in sulfate uptake and/or translocation. They are localized to the plasma membrane, with members of group 3 also localized to the chloroplast envelope (Cao et al., 2013; Takahashi et al., 2000; Yoshimoto et al., 2002, 2003). SULTRs of group 4 (SULTR4) are localized to the tonoplast and remobilize sulfate from the vacuole into the cytosol (Kataoka et al., 2004). *SULTR1* and *SULTR4* genes appear to be present throughout the green lineage (*Viridiplantae*), as they have also been identified in the basal plants *Physcomitrella patens* (bryophyte) and *Selaginella moellendorffii* (lycophyte) and in algae species, such as

*Chlamydomonas reinhardtii* (Kopriva et al., 2007; Takahashi et al., 2012).

In Arabidopsis, the genes *AtSULTR4;1* and *AtSULTR4;2* encode for tonoplast-localized transporters (Hawkesford, 2003). A recent structural analysis of *AtSULTR4;1* demonstrated that it forms a homodimer acting as a $H^+/SO_4^{2-}$ symporter. Each subunit consists of 14 transmembrane helices followed by a STAS (Sulfate Transporter and Anti-Sigma Factor Antagonist) domain, which contributes to the stabilization of the homodimer and regulates the sulfate transport activity (Wang et al., 2021). *AtSULTR4;1* and *AtSULTR4;2* transporters have been shown to mediate vacuolar sulfate remobilization in the root vasculature to facilitate the delivery of sulfate to the xylem and increase its transport to the shoot, especially under S deficiency (Kataoka et al., 2004). *AtSULTR4;1* is also expressed in developing Arabidopsis seeds, where it may remobilize sulfate for metabolic adjustments (Zuber et al., 2010). In *Bras-sica napus*, both *BnSULTR4;1* and *BnSULTR4;2* are up-regulated in response to S limitation in old or middle-aged leaves (Dubousset et al., 2009; Parmar et al., 2007), where they can unload the vacuolar sulfate store to sustain the S demand of proliferating cells in young leaves under S limitation (Dubousset et al., 2009). *SULTR4;1* is also up-regulated in the leaves of the model legume plant *Medicago truncatula*, when S deficiency occurred at the beginning of flowering. Since seed yield was not affected under that condition, *MtSULTR4;1* has been proposed to support the high demand of the younger tissues, including seeds, for sulfate (Zuber et al., 2013). However, no study has investigated the physiological significance of vacuolar sulfate remobilization during the reproductive period. Here, we aim to provide an understanding of the role of vacuolar sulfate transporter *SULTR4* for the production and nutritional quality of pea seeds. Therefore, we identified by TILLING (Targeting Induced Local Lesions IN Genomes) a series of allelic mutations in the only gene encoding a vacuolar sulfate transporter, hereafter called *PsSULTR4*, in "Caméor" (Dalmais et al., 2008), a high-protein pea cultivar whose genome has been recently sequenced (Kreplak et al., 2019). The functional analysis of these mutants shed light on the critical role of *PsSULTR4* in balancing sulfate homeostasis to maintain seed yield and seed quality under variable S supply.

## RESULTS

### PsSULTR4 is the only vacuolar sulfate transporter in pea

A phylogenetic analysis of the SULTR protein sequences in pea and Arabidopsis revealed that there is only one gene (*Psat3g185920*) belonging to group 4 of SULTR transporters in the pea genome (Figure 1A). The protein encoded by this gene, called *PsSULTR4*, shares 77.22 and 69.96% residue identity with *AtSULTR4;1* and *AtSULTR4;2*, respectively. To confirm the localization of *PsSULTR4* in
the tonoplast, PsSULTR4 fused to the red fluorescent protein (PsSULTR4-RFP) was transiently expressed in tobacco leaves under the control of the Arabidopsis ubiquitin-10 promotor (At4g05320, pUBQ10 from Grefen et al., 2010). The PsSULTR4-RFP signal in pavement cells (i.e., red signal in Figure 1B) appeared characteristic of the tonoplast of the central vacuole, which was adjacent to the nuclei, as revealed using DAPI (4',6-diamidino-2-phenylindole, cyan signal in Figure 1B), and was characterized by transvacuolar strands (see the enlarged panel in Figure 1B). The tonoplast localization was independently confirmed by fusing PsSULTR4 to the green fluorescent protein (PsSULTR4-GFP) and observing it in mesophyll cells (Figure S1).

The finding of only one SULTR4 copy in pea prompted us to investigate the number of *SULTR4* copies among 15 legume species belonging to different tribes (Figure S2). Only one *SULTR4* gene was identified in these species, except for soybean (*Glycine max*), lupin (*Lupinus angustifolius*), and the cultivated peanut (*Arachis hypogaea*), whose genomes have undergone polyploidy events (Figure S2). It is interesting to note that no more than two copies were identified, regardless of genome evolution events: duplications for soybean (Schmutz et al., 2010; Zhuang et al., 2019), triplication for lupin (Cannon et al., 2015), and hybridization of two diploid wild species, *Arachis ipaensis* and *Arachis duranesis*, leading to the allotetraploid cultivated peanut (Zhuang et al., 2019).

Next, we aimed to predict the protein structure of PsSULTR4 using AlphaFold (Jumper et al., 2021; Varadi et al., 2022). To test the reliability of this predictive tool for SULTR4-type proteins, the AlphaFold-simulated structure of AtSULTR4;1 was compared to its experimentally determined structure (residues 70–644) (Figure S3) (Wang et al., 2021). The root mean square deviation (RMSD) between the backbone heavy atoms of the two overlaid structures is 1.686 Å (Figure S3), demonstrating substantial similarity between both structures. Based on the high accuracy of the prediction, AlphaFold was used to predict PsSULTR4 structure from amino acid residues 64–638, corresponding to amino acid residues 70–644 of AtSULTR4;1 (Figures 1C and 2A). The overlay of the predicted structure of PsSULTR4 with the resolved structure of AtSULTR4 uncovered a remarkable degree of conservation (RMSD = 1.692 Å) (Figure 1D). PsSULTR4 consists of 12 transmembrane domains potentially embedded in the tonoplast, a vacuolar part, and a C-terminal region that includes the cytosol-localized STAS domain consisting of 125 amino acid residues (Figures 1C and 2A).

## Identification and molecular characterization of pea TILLING mutants for PsSULTR4

To address the physiological relevance of sulfate remobilization from the vacuole in the pea crop, we applied TILLING to identify mutated alleles of PsSULTR4. A screening

of the TILLING library in the “Caméor” background was performed by targeting two different regions of the gene, the first two exons, and exons 14–17 corresponding to the STAS domain. Five TILLING lines harboring point mutations predicted to affect protein function based on PROVEAN scores (Choi & Chan, 2015) were selected for phenotyping. This allelic series consisted in a nonsense mutation introducing a premature stop codon at position 78 (W78*) and four missense mutations. The missense mutations were all located in the STAS domain, changing aspartic acid at position 540 to asparagine (D540N), glutamic acid at position 568 to lysine (E568K), arginine at position 593 to tryptophan (R593W), and serine at position 613 to phenylalanine (S613F) (Figure 2). To assess the conservation of these four residues (D540, E568, R593, S613), an amino acid sequence alignment of SULTR4 from 21 representative species of each major lineage of land plants (Table S1), including pea and Arabidopsis, was performed (Figure 2B; Figure S4). Only E568 appeared strictly conserved across all species, suggesting the importance of this position for transport activity (Figure 2B). The Alpha-Fold prediction of PsSULTR4 structure after replacing the glutamic acid at position 568 with a lysine suggests that this amino acid change does not alter the structure of the protein (Figure S5). However, the E residue is negatively charged, while the K residue is positively charged (Figure S5). The E568K mutation may, therefore, influence the electrostatic property of the STAS domain rather than the folding of the protein. Whether the other missense mutations can influence the structure of PsSULTR4 has also been tested with AlphaFold, but none showed significant structural alterations (Figure S5). However, it cannot be excluded that these mutations alter the protein stability.

Next, the impact of the mutations on the synthesis or stability of the PsSULTR4 transcripts was studied in the leaves of young pea plants from the five homozygous mutants and their corresponding wild-type siblings (Figure 2D). A sharp reduction of PsSULTR4 transcript accumulation was observed for the nonsense W78* mutant, strongly suggesting that the premature stop codon caused nonsense-mediated decay of the mRNA. Transcript abundance remained unchanged for the mRNA variants D540N, E568K, and S613F. The steady-state level of the R593W transcript was decreased compared to its segregating wild type, since the SULTR4 mRNA level in this segregating wild type was significantly enhanced (Figure 2D).

## PsSULTR4 allows the maintenance of seed yield by remobilizing stored sulfate under S deficiency

To study the effect of the mutations in PsSULTR4 in planta, the five homozygous mutants and their wild-type siblings were grown under two conditions. In the S-adequate condition (+S), pea plants were continuously irrigated with a solution containing 0.3 mm sulfate. In the S-deficient
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**Figure 1.** Identification of Psat3g185920 as the only sulfate transporter of group 4 encoded in the pea genome.

(A) Phylogenetic tree based on the protein sequences of the sulfate transporters (SULTRs) of groups 1-4 from Arabidopsis and pea (1000 bootstrap replicates) (Kreplak et al., 2019) (The Arabidopsis Genome Initiative, 2000, https://doi.org/10.1038/35048692).

(B) Localization of PsSULTR4 in tobacco cells after transient expression of PsSULTR4-RFP in tobacco leaves (Bars = 20 µm). Representative images showing the Differential Interference Contrast (DIC), PsSULTR4-RFP signal (red), and DAPI signal (nuclei staining, cyan) of a transfected pavement cell are presented. The last image depicts an enlarged section (dashed box in previous images) showing the overlay between the PsSULTR4-RFP and DAPI channels. The arrow shows a transvacuolar strand (Reisen et al., 2005), and the arrowheads highlight the region around the nuclei (DAPI signal, cyan) representing the tonoplast labeled with SULTR4-RFP (red) and the absence of signal on the side of the nucleus facing the cell surface. DAPI, 4',6-diamidino-2-phenylindole.

(C) Prediction of Psat3g185920 (PsSULTR4) monomer structure with the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/). Data are visualized with the PyMOL software (v2.5.4) and show PsSULTR4 residues 64 to 638. The STAS domain of PsSULTR4 is indicated in green. The intracellular localization of SULTR4 proteins (left side) is based on data shown in Wang et al. (2021).

(D) The predicted structure of PsSULTR4 (residues 64–638, turquoise) was superimposed with the AtSULTR4;1 dimer (gray), whose structure has been experimentally determined by cryo-electron microscopy (PDB code = 7LHV, https://www.rcsb.org/structure/7LHV, Wang et al., 2021). Root mean square deviation = 1.692 Å. Sulfate molecules resolved in the AtSULTR4;1 structure are represented as a calotte model (yellow and red atoms, red arrow).

condition (–S), pea plants were irrigated with the same solution during the vegetative phase but deprived of sulfate (0 mm sulfate) throughout the reproductive period. Under +S, all *sultr4* mutants produced similar amounts of seeds than the wild-type lines (Table S2). In contrast, the W78* and E568K mutations caused a sharp and significant decrease in seed yield (i.e., total seed weight per plant) when plants are deprived of S (Table S2). The results obtained for these two mutants were confirmed in an independent experiment in which the seed yield of W78* and E568K *sultr4* mutants was decreased by 40 and 30%, respectively, compared to their wild type under –S (Figure 3A; Table S3). In both mutant lines, the decrease in seed yield was caused by a lower number of seeds, while individual seed weight was unaffected (Figure 3B,C; Table S3). It is worth noting that S deficiency during the reproductive phase did not affect the seed yield of the wild type (Figure 3A; Table S3). These results indicate that PsSULTR4 enables wild type pea to maintain seed yield in the absence of exogenous S during the reproductive period. Due to the substantial impact of the W78* and E568K mutations on the yield under S deficiency, we selected these mutants for further analyses.

We first analyzed the impact of the mutations in PsSULTR4 on the ability of seeds to accumulate S, nitrogen (N), and carbon (C). To this end, the concentration of S, C, and N in the mature seed compartment was determined, and the total quantity of C, N, and S per seed (i.e., in one seed) was calculated (Table S4). In all genotypes, S deficiency decreased the S content and quantity per seed compared to seeds produced upon sufficient S supply (Figure 3D; Table S4) but did not significantly affect quantities of C and N (Figure S6; Table S4). Remarkably, the W78* and E568K mutations in PsSULTR4 did not impact seed S, N, and C concentration and amount per seed, compared to the wild types, in either –S or +S conditions (Figure 3D; Figure S6; Table S4), demonstrating that S loading into seeds was not significantly affected by the *sultr4* mutations.

Next, we investigated the extent to which S partitioning between plant parts was affected in the mutants grown

under S deficiency, which reduced seed yield. This analysis was carried out on mature plants (Figure 3E) and on plants entering the early stage of the reproductive period (12 days after the beginning of flowering [DAF], Figure 3F) by measuring S content in all plant compartments. Then, we calculated the total amount of S in each compartment (S content multiplied by tissue biomass; all data are available in Table S5) and expressed it as a percentage of total S in the plant (Figure 3E,F). At both developmental stages, the mutants allocated significantly less S to the upper parts (seeds/pods and leaves developed during S deficiency, from node 9) than the wild types (Figure 3E,F). The amount of S in vegetative leaves (lower leaves developed before S deficiency, 1–8 nodes) was significantly higher in both *sultr4* mutants, with no significant change in the biomass of this plant compartment and in the total number of nodes at the mature stage (Table S6). To determine whether the defect in S allocation to the upper parts under –S is caused by decreased sulfate utilization, sulfate concentration was measured in vegetative leaves from nodes 6 to 8 at the mid-stage of the reproductive period (25 DAF). Remarkably, the sulfate concentration in these leaves was significantly higher in both *sultr4* mutants compared to the wild type (Figure 3G). We infer from these findings that PsSULTR4 enables the remobilization of the vacuolar sulfate stored in vegetative leaves to support S allocation to reproductive parts and maintain seed production when soil S is limiting.

### PsSULTR4 allows the utilization of stored sulfate in the endosperm of developing seeds

The impact of PsSULTR4 mutations on the accumulation of sulfate in mature pea seeds was investigated by using the same seed samples used for S content determination (Figure 3D). S deprivation in the reproductive period lowered the sulfate concentration of mature seeds derived from the wild type and *sultr4* mutants (Figure 3H). Interestingly, *sultr4* seeds developed under +S condition accumulated significantly higher sulfate levels than the wild type (W78*: 2.4-fold, E568K: 2.7-fold) (Figure 3H). Since total S loading in seeds was not affected in both mutants
(Figure 3D), this result indicates a substantial defect in sulfate assimilation in *sultr4* seeds well supplied with S. Under -S condition, although *PsSULTR4* expression was up-regulated in developing pea seeds (Figure S7), such an increase in sulfate concentration was absent in the mature

seeds of the *sultr4* mutants, which can be explained by the low availability of sulfate in the seeds compared to +S condition (Figure 3H). Furthermore, the expression level of *PsSULTR4* was three times higher in early developing seeds than in leaves harvested in parallel on the same day

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**Figure 2.** Characterization of TILLING mutations in PsSULTR4 and their effect on PsSULTR4 mRNA level.

(A) Protein sequence of PsSULTR4. The five residues for which mutations were identified by TILLING are shown in red and labeled by a red triangle. The 12 putative transmembrane domains (orange) of PsSULTR4 were predicted by TMHMM and refined using experimental evidence obtained in Wang et al. (2021). The protein sequence encoding the STAS domain is shown in blue. The pink labels indicate the beginning and the end of the corresponding sequence in AtSULTR4;1, whose structure has been resolved by cryo-electron microscopy (Wang et al., 2021).

(B) The STAS domains of SULTR4 proteins from 21 plant species (including PsSULTR4, bold) were aligned with the BioEdit software v7.0.9 (Hall, 1999). The selected species were representative of each major land plant lineage, according to the APG VI classification (The Angiosperm Phylogeny Group, 2016). The species, their taxonomy, and alignment-related data are detailed in Table S1, and the alignment of the entire SULTR4 sequences is shown in Figure S4. Phylogenetic relationships between the species are represented by the tree on the left, and taxonomic groups are indicated by the colored marks. Secondary structure domains (beta-strands by arrow, alpha-helices by helices) are indicated above the protein sequence. The mutated residues identified by TILLING in the STAS domain of PsSULTR4 are indicated by a red triangle. The amino acid exchange induced by TILLING is shown below the triangle. Identical and similar residues are highlighted in yellow and green, respectively.

(C) Diagram of the predicted topology of PsSULTR4 constructed using TMRPres2D 0.91 (biophysics.biol.uoag/TMRPres2D/, Spyropoulos et al., 2004), including the position of the five TILLING mutations (red dots).

(D) Relative transcript level of PsSULTR4, as determined by qRT-PCR, in leaves of 6-week-old *sultr4* mutants and wild-type plants deprived of S for 6 days. The graph shows means ± SD of data obtained from five to six individual plants. Statistically significant differences (*P* < 0.05) between all genotypes are indicated by different letters (ANOVA followed by Tukey's multiple comparisons test).

(9 days after flowering) under +S condition (Figure S7). Based on these findings, we hypothesized that under +S conditions, the PsSULTR4 transporter is critical for remobilizing vacuolar sulfate in seeds, allowing efficient sulfate assimilation into SAA during seed development. This hypothesis prompted us to study the kinetics of PsSULTR4 expression during seed development in the "Caméor" cultivar grown under +S. The relative expression level of PsSULTR4 was measured by microfluidic quantitative PCR (qPCR) in the same seed samples as those used to study the kinetics of SSP synthesis in "Caméor" (Kreplak et al., 2019). PsSULTR4 expression peaked at the transition between embryogenesis and seed-filling stages (12 days after pollination [DAP]). Although weaker after this stage, its expression remains stable during the period associated with the synthesis of SSP belonging to the albumin (PA1) and globulin (vicilin and legumin) families (up to 23 DAP) (Figure 4A). The relative expression level of PsSULTR4 in the three seed tissues (seed coat, endosperm, and embryo) was then evaluated by reverse transcription quantitative PCR (RT-qPCR) at the 12 DAP stage. Its expression was more than 10-fold higher in both the seed coat and endosperm when compared to the embryo (Figure 4B), suggesting that PsSULTR4 could play an important role in the tissues surrounding the embryo.

To test this hypothesis, sulfate concentration was measured in the separated tissues of the wild-type and the *sultr4* seeds collected at 12 DAP. Interestingly, sulfate concentration in the endosperm of *sultr4* mutant seeds was about nine times higher than in wild-type seeds (Figure 4C). Since the fresh weight of each of the three tissues was similar between the genotypes (Figure 4D), this substantial increase is not caused by altered endosperm development in *sultr4* mutants. These data suggest that PsSULTR4 allows the stored sulfate in the endosperm to be remobilized at the transition to seed filling and presumably used by the embryo as a source of S for storage protein deposition.

PsSULTR4 contributes to the accumulation of S-rich storage proteins in seeds

To provide further evidence for the impaired sulfate assimilation in *sultr4* seeds, the SSP composition of mature seeds of the W78* and E568K mutants and wild types, grown under both S nutrition conditions, was determined by Fast Performance Liquid Chromatography (FPLC). This method allows the separation of PA1 and PA2 albumins, 7S globulins, and 11S globulins, followed by their quantification based on the area under the peak of the detected SSP (Table S7). In response to S deficiency, wild-type seeds accumulated less S-rich PA1 albumins and more S-poor 7S globulins (Figure 5A–C). Interestingly, under +S condition, both *sultr4* mutants accumulated lower amounts of the PA1 albumins, which are the SSP with the highest SAA content in their sequence (~10%) (Figure 5D,E). This decrease was compensated by an increase in S-poor 7S globulins (Figure 5F), which have less than 1% SAA in their sequence, explaining how the amount of C and N per seed was maintained in *sultr4* seeds (Table S4). It is worth noting that although the *sultr4* mutants were well supplied with S, this adaptation is the typical response of wild-type seeds to S deficiency (Figure 5A–C). It should be noted that in mutant seeds developed under –S, the relative abundance of PA1 albumins and 7S globulins did not change significantly compared to the wild types (Figure 5G–I). The only significant difference was for PA2 albumins, which decreased in the W78* mutant seeds compared to wild-type seeds but not in the E568K mutant seeds (Table S7). These results support a prominent role for PsSULTR4 in determining the protein composition of seeds well supplied with S and strongly suggest that the balance between the S-rich PA1 albumins and the S-poor 7S globulins depends not only on seed S uptake but also on the remobilization of vacuolar sulfate in the endosperm.

Our study demonstrates that PsSULTR4 is critical at two levels for whole plant S use efficiency: (i) PsSULTR4
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<caption>**Figure 3.** Impact of SULTR4 mutations on seed production, allocation of sulfur during the reproductive period, and seed sulfur content.</caption>

(A) Seed yield of *sultr4* mutants and their corresponding wild types (WT1 for W78* and WT2 for E568K) grown under S-sufficient (+S) and S-deficient (−S) conditions (data are from 6 to 10 individual plants).

Number of seeds (B) and one seed weight (C) for *sultr4* mutants and their corresponding wild types grown under −S (6 < *n* < 10).

(D) S quantity per seed estimated for the *sultr4* mutants and their corresponding wild types (WT1 for W78* and WT2 for E568K) grown under +S and −S conditions (*n* = 6–10 individual plants).

(E, F) Partitioning of total S between tissues of the *sultr4* mutants and their corresponding wild types grown under S deficiency (−S) at the mature stage [(E), *n* = 6–10] and 12 days after flowering [(F), *n* = 8, DAF]. The identities of tissues are indicated by a color code defined in the boxed legend. The data are expressed in percentage of total plant S, with mean values of S amount in each tissue indicated by white numbers (in mg, on a dry weight basis) (data obtained for individual plants are available in Table S5). Mean values (±SD) of the total S amount (in mg, on a dry weight basis) of the respective genotype is shown in black above the stack plots. At the 12 DAF stage, the plants endure 17 days of S deficiency. N1–N8 corresponded to leaves developed before S deficiency (i.e., lower leaves), and N9-top corresponded to the leaves developed after S deficiency to the top of the plant (i.e., reproductive leaves). At the mature stage, the seeds were separated from the pod walls, whereas at the 12 DAF stage, the pods containing the seeds were used for analysis.

(G) Sulfate concentration in vegetative leaves of plants grown under −S (25 DAF corresponds to seed filling of the first pods, *n* = 4).

(H) Sulfate concentration in seeds of *sultr4* mutants and their corresponding wild types (WT1 for W78* and WT2 for E568K) grown under +S and −S conditions (*n* = 4 individual plants). Data are presented as the mean ± SD in all panels. Statistically significant differences revealed by the Tukey's test (*P* < 0.05) are indicated by different letters [significant differences between all samples, panels (A–D, G–H)] or asterisks [significant difference between the mutant and its corresponding wild type, panels (E, F)].

<figure><img src="image_18.png" /><figcaption>**Figure 4.** Expression pattern of *PsSULTR4* during seed development, between seed tissues, and effect of the mutations on sulfate in seeds developed under S sufficiency.</figcaption></figure>

(A) Relative expression levels of *PsSULTR4* (black bars), 2S albumin PA1 (Psat1g073600, yellow line), 7S globulin (Vicilin gene, Psat0s5g0160, blue line), and 11S globulin (legumin gene, Psat3g058800, green line) during pea seed development as determined by microfluidic qRT-PCR, starting 8 days after pollination (DAP, embryogenesis stage) till 29 DAP (late maturation stage) (*n* = 3). The data for storage protein genes are taken from Kreplak et al. (2019).

(B) Relative expression level of *PsSULTR4* (qRT-PCR) in the seed coat, the endosperm, and the embryo of wild-type seeds (12 DAP developmental stage) grown under S sufficiency (cv. Caméor, *n* = 3).

(C) Sulfate concentration in the seed coat, the endosperm, and the embryo of *sultr4* mutants and their corresponding wild types (WT1 in front of W78* and WT2 in front of E568K) grown under S-sufficient (+S) condition. The seeds were collected at the 12 DAP stage (*n* = 3, tissues of ≈20 seeds for each replicate).

(D) Fresh weight of the seed coat (light green), the endosperm (yellow), and the embryo (dark green) of *sultr4* mutants and their corresponding wild types (WT1 for W78* and WT2 for E568K) grown under S-sufficient (+S) condition (seeds were collected at 12 DAP from 3 to 4 individual plants). Data are presented as means ± SD. In each graph, different letters indicate significant statistical differences (*P* < 0.05) between sample groups (ANOVA followed by Tukey's multiple comparisons test).
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**Figure 5.** Contribution of PsSULTR4 to seed storage protein composition.

(A–C) Storage protein composition of mature seeds from wild-type pea plants grown under S sufficiency (+S) and S deficiency (−S) as determined by fast performance liquid chromatography. (A) Superimposition of representative chromatograms obtained from wild-type seeds grown under +S (black line) and −S (red line). Peaks corresponding to the major SSP (PA2, orange; PA1, yellow; 7S globulins, blue, and 11S globulins, green) are labeled above the respective peak. Colored arrows indicate changes of SSP triggered by S deprivation. (B, C) The relative abundance (in % of total peak area) of the S-rich PA1 albumins (B) and the S-poor 7S globulins (C) in seeds of the wild type grown under +S (dark blue) and −S (orange). Arrows indicate the shift induced by −S in WT1 and WT2.

(D–F) Storage protein composition of mature *sultr4* and wild-type seeds from plants grown under +S. (D) Superimposition of representative chromatograms obtained from wild type (dark blue) and *sultr4* W78* and E568K seeds (red) grown under +S condition. (E, F) The relative abundance (in % of total peak area) of the S-rich PA1 albumins (E) and the S-poor 7S globulins (F) in seeds of the wild-type (dark blue) and *sultr4* mutants (red).

(G) Superimposition of representative chromatograms obtained from wild-type (dark blue) and *sultr4* W78* and E568K seeds (red) grown under −S condition.

(H, I) The relative abundance (in % of total peak area) of the S-rich PA1 albumins (H) and the S-poor 7S globulins (I) in seeds of the wild-type (dark blue) and *sultr4* mutants (red). Arrows indicate the shift induced by mutations of PsSULTR4. Data in panels (B, C, E–H) are presented as means ± SD of 4–6 biological replicates of seeds (i.e. from individual plants). In each panel, different letters indicate statistically significant differences (*P* < 0.05) between the sample groups (ANOVA followed by Tukey's multiple comparisons test).

remobilizes stored vacuolar sulfate in source tissues to maintain seed production when external S availability is low; (ii) PsSULTR4 fine-tunes sulfate remobilization in the endosperm as a critical control point for storage activities in seeds well supplied with S.

DISCUSSION

In this study, we investigated the role of PsSULTR4 in seed yield and quality establishment in the legume crop pea, whose cultivation and use represent a lever for accelerating the agroecological and food transitions. First, we have shown that the genome of many legume species, including pea, contains only one copy of the *SULTR4* gene and that genome evolution events have resulted in a maximum of two copies of this gene (Figure S2). This is the case for soybean, whose genome has been duplicated twice, and for lupin, whose genome has undergone one triplication (Cannon et al., 2015; Schmutz et al., 2010; Zhuang et al., 2019). The retention mechanisms of two copies may result from gene dosage balance, neofunctionalization, or subfunctionalization (Gout & Lynch, 2015). In the Arabidopsis genome, which has undergone duplication events (Bowers et al., 2003; Vision et al., 2000), two copies, *AtSULTR4;1* and *AtSULTR4;2*, have been retained (Figure 1A). Both genes are predominantly expressed in the vasculature of the seedling root and hypocotyl (Kataoka et al., 2004). *AtSULTR4;1* is constitutively expressed in the root regardless of S availability, whereas *AtSULTR4;2* is preferentially expressed in this tissue under S deficiency, suggesting a partial functional redundancy between these two genes. Under low S availability, vacuolar sulfate efflux allows S distribution from the root to the shoot, with AtSULTR4;1 playing a major role and AtSULTR4;2 a supplementary function (Kataoka et al., 2004). Furthermore, both genes are expressed during Arabidopsis seed development, with *AtSULTR4;1* exhibiting higher expression levels than *AtSULTR4;2* (Zuber et al., 2010).

The amino acid sequence of SULTR4 is well conserved among land plants (Figure S4), and the predicted protein structure of PsSULTR4, the only SULTR4 transporter in the pea genome, is very similar to that experimentally

determined for AtSULTR4;1 (Figure 1C,D) (Wang et al., 2021). The high sequence and structural homology of the STAS domain (Figure 2B) reflects its essential function in plants, as described in Arabidopsis, where several point mutations in this domain have been shown to affect sulfate transport activity (Shibagaki & Grossman, 2004, 2006; Wang et al., 2021). In our study, we have shown that mutation of the strictly conserved residue E568 of the STAS domain of PsSULTR4 (E568K substitution) drastically affects the utilization of the vacuolar sulfate store in both leaves and seeds of pea to a similar extent as the W78* nonsense mutation (Figures 3 and 4), supporting a role for this glutamate residue in the PsSULTR4 function. This also applies to plasma membrane-localized SULTR, since the same substitution in the same glutamate residue (E578K, corresponding to E568K in PsSULTR4) impairs the accumulation of AtSULTR1;2 in the plasma membrane of yeast. This likely explains the lack of rescue of the SULTR-deficient yeast strain CP154-7B by AtSULTR1;2E578K expression (Shibagaki & Grossman, 2006). As this mutation changes the charge of the residue and is located in one of the beta strands (Wang et al., 2021; also highlighted in Figure 2B), it may alter the organization/stabilization of the β-sheet, although no noticeable change was observed in the predicted structure of the STAS domain carrying this mutation (Figure S5).

Characterization of the pea *sultr4* mutants E568K and W78* revealed that by enabling the utilization of the sulfate store in lower leaves, PsSULTR4 triggers the distribution of S to reproductive tissues (leaves of flowering nodes, pods, and seeds), allowing pea to produce the standard number of seeds despite S depletion during the reproductive period (Figure 3). Lower but non-negligible amounts of S were detected in reproductive leaves and seeds of the *sultr4* mutants subjected to S deficiency (Figure 3E,F). This could be explained by the ability of plants to transport other S molecules from source to sink tissues, such as S-methylmethionine and glutathione, which can be used for the synthesis of Met and Cys, respectively (Bourgis et al., 1999; Gallardo et al., 2007; Noctor et al., 2012). Although PsSULTR4 allows for the maintenance of seed
yield under S deficiency, this is at the expense of the protein quality of seeds. The seeds contained low amounts of S-rich PA1 albumins, which were compensated by high amounts of S-poor 7S globulins (Figure 5A–C). The data obtained under the –S condition showed no significant changes in the relative abundance of these proteins between the wild type and *sultr4* seeds (Figure 5H,I), which contain a low concentration of sulfate (Figure 3H). Therefore, although *PsSULTR4* was found to be up-regulated in response to –S in developing seeds (Figure S7), we concluded that vacuolar sulfate remobilization plays a prominent role in maintaining seed production rather than seed protein quality under S deficiency, which limits sulfate accumulation in seeds. The importance of the delivery of sufficient amounts of S coming from the phloem to developing seeds for protein quality in legumes has been shown in several studies (Garneau et al., 2021; Tabe & Droux, 2002). Overall, our data indicate that S fertilization during the vegetative period would be sufficient to maintain seed yield in pea because of the efficient vacuolar sulfate remobilization by PsSULTR4. However, prolonged S fertilization during the reproductive period is beneficial to maintain the accumulation of S-rich SSP, that is, the 11S globulins and 2S albumins, which enhances the nutritional value of legume seeds and, consequently, seed quality (Duranti & Gius, 1997).

One of the most striking observations in this study was the decreased seed protein quality of the *sultr4* mutants grown under +S condition. Mature seeds of the E568K and W78* mutants contained lower amounts of the S-rich PA1 albumins than wild-type seeds (Figure 5D–F), mirroring what happens in wild type subjected to S deficiency (Figure 5A–C), even though the amount of total S in seeds was unchanged (Figure 3D). Significantly, the *sultr4* mutant seeds contained higher concentrations of sulfate compared to the wild type under +S condition (Figure 3H). The same observation was made in a previous study on AtSULTR4;1 (*sultr4*;1 T-DNA mutants), where a role for this transporter in mediating the utilization of the vacuolar sulfate pool within the seed was proposed (Zuber et al., 2010). The results obtained in the present study additionally revealed that under S deficiency, *PsSULTR4* expression peaks at the transition stage between embryogenesis and seed filling (12 DAP). This expression remains relevant for a sustained period associated with storage protein synthesis (up to 23 DAP) and declines sharply once late maturation is initiated (Figure 4A,B). Interestingly, we demonstrated that PsSULTR4 mediates sulfate utilization in the endosperm, with about ninefold more sulfate retained in the *sultr4* mutant endosperm compared to the wild type (Figure 4C). In dicotyledons, the endosperm is crucial in providing nutrients to the filial tissue (Baud et al., 2008). While the mechanisms underlying S transport from the endosperm to the embryo remain elusive, our results suggest that the

endosperm can supply the seed embryo with sulfate at key stages of seed development associated with the onset of SSP synthesis (Figure 4A). In support of this hypothesis, a metabolic role of the endosperm as a source of assimilates for storage activities in the embryo has been demonstrated in peas (Melkus et al., 2009). Indeed, these authors have shown that during early seed development, when the sink strength of the embryo is low, the endosperm stores excess nutrients such as glutamine to be released into the embryo via active system transports once seed filling is initiated. Our results suggest that sulfate may be one of these nutrients. This is reinforced by a study showing that enhancing S loading in the embryo of pea seeds increased sulfate levels in the endosperm, along with the positive regulation of sulfate transporters and reduction pathways in the embryo (Garneau et al., 2021).

Based on the data obtained in this study, we propose a model explaining the role of PsSULTR4 in the trade-off between seed yield and seed protein quality to ensure optimal utilization of the vacuolar sulfate store (Figure 6). When seed set and development occur under –S condition, PsSULTR4 prioritizes sulfate distribution from vegetative tissues to the reproductive parts of pea plants to maintain the number of seeds per plant (Figure 6A,B). This limits sulfate accumulation in the individual seeds, reducing the relative abundance of S-rich seed proteins, which is compensated by an enhanced synthesis of the S-poor proteins to maintain seed N content. In contrast, when pea plants are well supplied with sulfate throughout their life cycle, PsSULTR4 plays a more prominent role in the establishment of seed protein quality by remobilizing sulfate that has been stored within the seed for the accumulation of SSP rich in SAA, which are essential for human nutrition (Figure 6C,D). Our data also showed that sulfate in the seed endosperm during embryogenesis and its release by PsSULTR4 at the onset of filling is likely to be a decisive control point in coordinating storage activities in the pea embryo. Our findings open new perspectives for understanding the intricate communication between seed tissues regarding sulfate transport and metabolism in relation to storage protein synthesis in legume seeds.

## EXPERIMENTAL PROCEDURES

### Alignments and phylogenetic trees

The amino acid sequences of all Arabidopsis SULTRs were downloaded from the TAIR database (https://www.arabidopsis.org/), The Arabidopsis Genome Initiative, 2000). The pea SULTRs were identified with the BLASTp tool (https://urgi.versailles.inra.fr/blast/, E-value = 0) using the amino acid sequence of Arabidopsis SULTRs as templates and by selecting *Pisum sativum* genome V1a (Kreplak et al., 2019) as source. The amino acid sequence of SULTR4 in different legume species was obtained by blasting PsSULTR4 against sequences in the Legume Information System (LIS SequenceServer BLAST) (Priyam et al., 2019, https://
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Sulfur deficiency (-S)

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sultr4 mutant

Sulfur deficiency (-S)

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Sulfur sufficiency (+S)

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sultr4 mutant

Sulfur sufficiency (+S)

<caption>**Figure 6.** Proposed model for the role of PsSULTR4 in determining seed yield and protein quality in pea.

(A, B) Under S deficiency (–S), wild-type pea plant (A) remobilizes vacuolar sulfate in source tissues, especially vegetative leaves, by activating SULTR4 to ensure high seed yield. However, due to limited S supply, the expression of S-poor SSP is favored, and the accumulation of S-rich SSP is decreased, resulting in low protein quality. In *sultr4* mutants (B), PsSULTR4 is not functional under –S. Consequently, sulfate remains blocked in the vacuole of vegetative leaves, resulting in a substantial decrease in seed yield.</caption>

(C, D) Under S sufficiency (+S), the wild-type pea plant (C) remobilizes vacuolar sulfate and uses external sulfate to produce the maximal number of seeds with a high content of S-rich storage proteins. Under +S conditions, *sultr4* mutants (D) can compensate for the lack of vacuolar sulfate remobilization by taking up more external sulfate. Thus, seed yield is unaffected, and sulfate substantially accumulates in vegetative leaves of mature *sultr4* plants. However, the lack of sulfate remobilization cannot be fully compensated since sulfate is trapped in the vacuole of endosperm cells in *sultr4* mutants. Consequently, mature seeds of *sultr4* mutants display, even under +S conditions, lowered accumulation of S-rich SSP and prefer to produce S-poor SSP. Due to the trapped vacuolar sulfate in the seeds of *sultr4* mutants, the total S content of *sultr4* seeds and wild-type seeds is indistinguishable.

sequenceserver.legumeinfo.org/). Trees were generated using the NGPhylogeny online tool (https://ngphylogeny.fr/) with 1000 boot-strap replicates, visualized using the iTOL version 6 tool (Letunic & Bork, 2021, https://itol.embl.de/), and graphically edited using the Inkscape software (version 1.2.1).

To study the conservation of the amino acid residues of SULTR4, 21 land plant species were selected as representative of each major land plant lineage from the APG IV classification (The Angiosperm Phylogeny Group, 2016): *Marchantia polymorpha* (NCBI:txid3197), *Ceratodon purpureus* (NCBI:txid3225), *Selaginella moellendorffii* (NCBI:txid88036), *Adiantum nelumboides* (NCBI:txid2759587), *Picea sitchensis* (NCBI:txid3332), *Nymphaea thermarum* (NCBI:txid419125), *Cinnamomum micranthum* (NCBI:txid337451), *Dioscorea alata* (NCBI:txid55571), *Zea mays* (NCBI:txid4577), *Papaver somniferum* (NCBI:txid3469), *Vitis vinifera* (NCBI:txid29760), *Glycine max* (NCBI:txid3847), *M. truncatula* (NCBI:txid3880), *Pisum sativum* (NCBI:txid3888), *Malus domestica* (NCBI:txid3750), *Citrus sinensis* (NCBI:txid2711), *Arabidopsis thali-ana* (NCBI:txid3702), *Spinacia oleracea* (NCBI:txid3562), *Camellia sinensis* (NCBI:txid4442), *Solanum lycopersicum* (NCBI:txid4081), and *Helianthus annuus* (NCBI:txid4232) (Table S1). The NCBI BLASTp search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search SULTR4 amino acid sequences from the available public database “All non-redundant GenBank CDS.” The sequence
with the best hit was retained for each selected species (E-value = 0). Phylogenetic relationships between these 21 species were rebuilt from the APG classification and written in a Newick formatted file. The iTOL version 6 tool (https://itol.embl.de/) was used to generate and edit the tree. Amino acid sequences were aligned using the BioEdit software (version 7.0.9, Hall, 1999) with the ClustalW alignment tool. The position of the STAS domain and secondary structure elements of all SULTR4 were based on AtSULTR4;1 data in the Protein Data Bank (PDB, id: 7LHV, doi: 10.2210/pdb7LHV/pdb, Wang et al., 2021).

## PsSULTR4 structure prediction

The resolved structure of AtSULTR4;1 was downloaded from the PDB database (PDB code: 7LHV). Formatted files, one with the PsSULTR4 amino acid sequence, and five with the sequence containing each of the five TILLING mutations, were used as input to run the AlphaFold Protein Structure Database at https://alphafold.ebi.ac.uk/ (Jumper et al., 2021; Varadi et al., 2022). The predicted protein structure for each of the proteins was downloaded as a PDB formatted file. The files were imported into PyMOL software (v2.5.4, from www.pymol.org, DeLano, 2002) for structural alignments and analyses based on RMSD (root mean square density) values.

## Transient expression of PsSULTR4 in tobacco leaves for subcellular localization

The Gateway technology was used for cloning according to the manufacturer's instructions (Invitrogen, Villebon-sur-Yvette, France). The PsSULTR4 coding sequence with the stop codon removed was amplified from Caméor cDNA by PCR using the primers PsSULTR4-Attb1-F and PsSULTR4-Attb2-R2 (Table S8). Amplification was performed using the Advantage DNA polymerase (Takara Bio Europe, Saint-Germain-en-Laye, France). The amplified product was cloned in the pDONR221 Gateway entry vector, then transferred in the ubiquitin-10 promoter-based vectors pUBQ10 containing the RFP or GFP coding sequences (Grefen et al., 2010; Nelson et al., 2007) to produce pUBQ10-PsSULTR4-RFP and pUBQ10-PsSULTR4-GFP. The positive clones (spectinomycin-resistant) were sequenced to confirm the identity with the corresponding PsSULTR4 coding sequence (universal primers M13F and M13R) and used to obtain Agrobacterium tumefaciens GV3101 transformants. Tobacco plants (Nicotiana tabacum "Xanthi") were grown for 4 weeks in a growth chamber with a 16-h photoperiod and day/night temperatures of 24-25°C/22-23°C and transiently transformed by infiltration of the leaf under epidermis, according to Sparkes et al. (2006). Constructs were infiltrated at an optical density at 600 nm of 0.3. Leaf samples (approximately 5 mm²) were observed for GFP or RFP fluorescence 4 days after infiltration. For nucleus-specific staining, the leaves were infiltrated with a 10 µg ml⁻¹ DAPI solution 30 min prior to observation (Sigma Aldrich, Saint-Quentin-Fallavier, France). Fluorescence observations were conducted with a Leica SP8 Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany) using the 40×/1.25 oil objective (Numerical Aperture = 1.25) and the LAS X Microscope Software (v3.7.9; Leica Microsystems). Excitation wavelengths and emission filters were 552 nm/band-pass 563-638 nm for RFP, 488 nm/band-pass 500-584 nm for GFP, 488 or 552 nm/band-pass 647-700 nm for chlorophyll, and 405 nm/band-pass 415-482 nm for DAPI.

## Screening of the "Caméor" TILLING population for PsSULTR4

Ethyl methanesulfonate-induced mutations in PsSULTR4 were identified by screening the TILLING population ("Caméor"

cultivar), consisting of 4800 M2 lines, according to the method described in Dalmais et al. (2008). Due to the complexity of the genomic sequence of PSULTR4;1 - Psat3g185920 (9761 kb and 17 exons), two amplicons were selected for the molecular TILLING screen. The first amplicon covered exons 1 and 2 (TILLING screening 1, Table S8), and the second amplicon covered exons 14-17 (TILLING screening 2, Table S8), corresponding to the STAS domain region. For each screening, a first fragment was amplified (PCR 1 in Table S8) and used as the template for the second PCR (PCR 2 in Table S8) with primers carrying a universal M13 tail (5'-CACGACGTTGTAAAACGAC-3' for the forward primer; 5'-GGATAACAATTTCACACAGG-3' for the reverse primer) labeled at the 5' end with infrared dyes IRD700 and IRD800 (LiCOR). To confirm mutations, PCR amplification products were sequenced (GATC Biotech, Konstanz, Germany) and analyzed with the Chromas v.2.6.6 software (Technelysium Pty., South Brisbane, Australia). Five mutations predicted to affect protein function according to PROVEAN scores (PROVEAN online tool, Choi & Chan, 2015, doi: 10.1093/bioinformatics/btv195, https://provean.jcvi.org/seq submit.php) were retained for this study. The five sultr4 mutants were backcrossed twice with the wild-type line "Caméor." All progenies BC1, BC2, BC1S1, and BC2S1 (selfing of BC1 or BC2) were genotyped with specific Caps or dCaps markers designed according to Neff et al. (1998). All primers and enzymes are listed in Table S8. For each mutation, all comparisons in this study were made between wild-type and mutant plants from the same progeny, following two backcrosses.

## Plant growth conditions and tissue collection

For phenotyping and harvesting tissues under S deficiency in parallel to S sufficiency, pea plants were grown in pots containing a mixture of perlite/sand (2/1, v:v), in a greenhouse (without S evaporator), at a day/night temperature of 18/14°C, with supplemental artificial lighting (16-h photoperiod, 250 µmol m⁻² sec⁻¹). The pots were irrigated with a nitrate- and S-rich nutrient solution containing 0.3 mM MgSO₄ (S-sufficient condition, +S) as described in Zuber et al. (2013). At a late vegetative stage (about 5 days before flowering, eighth node stage, 3 weeks-old plants), half of the plants from each genotype were subjected to S deficiency (−S) by rinsing the substrate twice with deionized water and twice with the −S solution in which MgSO₄ has been replaced by MgCl₂. In all −S experiments, S was deprived up to maturity of the plants. A preliminary experiment with the five TILLING mutants in segregation with their wild types was realized to assess seed production in both the +S and −S conditions (Table S2). Between two and five homozygous plants were identified by genotyping with Caps or dCaps markers (Table S8), and only the W78* and E568K mutants impacted seed production. A second experiment was carried out to assess seed yield and seed yield components (number of seeds and one-seed weight) with 6–10 replicates for the W78* and E568K mutants in each condition (Figure 3). The different compartments of the plants (roots, nodes 1–8, nodes 9 to the top, pods/seeds) were separated at two stages (12 days after the beginning of flowering and at maturity), and their dry weight was determined after oven-drying at 80°C for 48 h, except for dry mature seeds, which were weighed after oven-drying at 30°C for 24 h. For these experiments, the axillary buds were removed during plant growth, resulting in single-stemmed flowering plants. To study the impact of the five mutations on PsSULTR4 expression (Figure 2D), five to six plants per genotype were grown under the +S condition for 5 weeks, then S deficiency was applied for 6 days, until leaf collection.

For collecting developing seeds or seed tissues from the Caméor genotype under +S conditions, the same growth
conditions were applied except that the pot substrate was a mixture of vermiculite and clay balls (1:3, vol:vol), and that plants were automatically irrigated with a high inorganic N concentration solution referred to as the N+ solution in Guinet et al. (2018). For gene expression analyses during seed development (Figure 4A), emerging flowers from the second and third nodes were tagged on the day of pollination (Maurer et al., 1966), and labeled pods were harvested at 8, 12, 16, 19, 16, and 29 DAP stages. For each of the three biological replicates, seeds from two pods of the same stage were pooled, flash-frozen in liquid nitrogen, and stored at -80°C until extraction. In order to check that the seeds were at the same developmental stage, the water content of two seeds per pod was determined.

For gene expression analyses in the endosperm, seed coat, and embryo (Figure 4B), 12 DAP seeds obtained as described above were rapidly dissected manually on ice with a scalpel. The liquid endosperm of each seed was collected with a P10 pipette, pooled, and snap frozen. Testa and embryos were quickly rinsed twice into distilled water and gently blotted with Kimwipes® disposable wipers in order to remove any liquid endosperm from the surface and snap frozen. Three biological replicates were collected, with each sample consisting of about 20 individual seeds. The same procedure was used for sulfate measurements in the tissues of 12 DAP seeds (pool of 20 seeds per replicate) from the wild-type and TILLING mutants. Tubes were briefly immersed in liquid nitrogen and stored at -80°C.

## Gene expression analyses on pea leaves and seeds

Pea leaves and whole developing seeds stored at -80°C were ground in liquid nitrogen using a pestle and mortar. Embryos and seed coats from 12 DAP seeds were ground directly in 2 ml Eppendorf® tubes using a Retsch Mixer Mill 400 with stainless steel beads. Total RNA extraction of seed tissues was performed with the Spectrum™ Plant Total RNA Kit (Sigma Aldrich), and traces of genomic DNA were removed using the One-Column DNase Digest Set (Sigma Aldrich) following the manufacturer's instructions. For endosperm samples, the lysis buffer was added directly to the liquid endosperm, and the mixture was incubated for 3 min at room temperature before proceeding to the next step. Total RNA extraction of leaves was performed with the RNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France), including a DNase treatment with the RNase-Free DNase Set (Qiagen). RNA concentration and quality were evaluated using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Courtaboeuf, France). The RNAs were reverse-transcribed with the iScript cDNA synthesis kit (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer's protocol. Real-time qPCR (Figures 2d and 4b; Figure S7) was performed on a LightCycler 480 (Roche Diagnostics, Meylan, France) using the GoTaq qPCR Master Mix (Promega, Charbonnières-les-Bains, France) with 3 µl of diluted cDNA, 0.2 µl of each primer (10 µM) in a final volume of 10 µl, and the following program: 95°C for 5 min; and 40 cycles of 95°C for 10 sec and 60°C for 1 min. A melting curve analysis was performed by a further cycle from 50°C with a 0.11°C increase per second up to 95°C, and the fluorescence was measured every 0.5°C. Reactions were performed in duplicates from each biological replicate. The expression profiles of genes during seed development (Figure 4A) were obtained by high-throughput real-time qPCR using the Biomark microfluidic system from Fluidigm® (96.96 Dynamic Array™ IFCs, BMK-M-96.96) as described in Kreplak et al. (2019). In both cases, expression levels relative to the endogenous control genes were calculated using the ΔΔCT method (Schmittgen & Livak, 2008). Primer sequences of target and control genes are listed in Table S8.

## Measurements of S, C, N, and sulfate in plant tissue samples

S, C, and N contents were determined from dried, ground tissue samples from at least six biological replicates using the Dumas method (Allen, 1974) on a Carlo Erba elemental analyzer NC2500 (Thermo Fisher Scientific) adapted with a multi-separation column (polytérafluoroéthylène, 2 mm length, internal and external diameters of 5 and 6 mm, respectively; CE Elantech, Lakewood, New Jersey, https://www.ceelantech.com/). Prior to analysis, 2 mg vanadium pentoxide was added to 5 mg tissues.

The concentration of sulfate was determined from 30 mg of powdered dry mature seed samples and lower leaves according to the procedure described by Etienne et al. (2018). For lyophilized seed tissue powder, we applied the same extraction method with the following modifications due to the lower amounts of material (5 mg): 25 ml of 50% ethanol and ultra-pure water, the Integron HPIC system (Thermo Scientific-Dionex, Villebon-sur-Yvette, France) was used, the eluent solution for anion analysis consisted of 9 mM Na₂CO₃ and 20 µl was pumped isocratically over an analytical column (AS9-HC 4×250 mm). Anion concentrations were calculated using the Chromelon 7 software (version 7.2; Thermo Scientific-Dionex).

## Analysis of seed protein composition by FPLC

Seed proteins were extracted from 100 mg of mature seed powder pre-treated with acetone (400 µl/100 mg, 20 min shaking at 450 rpm) and centrifugated for 30 min (6000 g) at 4°C. The resulting pellet was air-dried overnight and resuspended in 2.5 ml extraction buffer (Tris 50 mM, K₂SO₄ 5%, EDTA 1 mM, β-mercaptoethanol 0.05%, benzamidine 1 µM, leupeptine 1 µM, PMSF 1 µM, pH 8.5) for 1 h on a shaker. The protein extract was centrifuged (6000 g, 20 min, 4°C) and filtered through 0.22 µm cellulose acetate syringe filters (Millipore Corp., Milford, MA, USA). FPLC was performed with a Waters (Waters Europe S.A.S., Saint Quentin en Yvelines, France) system (refs. 600E system controller, 717 injector, 486 detector) using a MonoQ 5/50 GL analytical column (Amersham Biosciences, Saclay, France). The elution buffer was Tris 50 mM, pH 8.5, and the NaCl gradient was 0-0.72 M (40 min, 1 ml min⁻¹). The detection was performed at 280 nm. Relative quantifications of FPLC fractions were obtained by integrating peak areas below the chromatogram for each fraction as follows. The raw data were processed using R 4.2.2 statistical software and the "ChemoSpec" library. Only chromatogram points between a retention time of 8 and 26 min were retained (removeFreq). Chromatograms were normalized (norm-Spectra) by redefining a baseline between points at 8 and 26 min, and the height of each peak was recalculated relative to this baseline (baseSpectra). The areas of all protein peaks were summed up and set to 100% and the relative percentage of specific fractions was calculated according to Bourgeois et al. (2011).

## Statistical analyses

Significant variations between sample groups (*P*-value <0.05) were determined by one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test, except for data in Figure S7, where a Student's *t*-test was applied for pairwise comparisons (*P* < 0.05 was considered statistically significant). All statistical analyses were conducted using the R Studio software (2022.07.1).

## ACKNOWLEDGMENTS

We would like to thank all the members of the 4PMI platform (Plant Phenotyping Platform for Plant and Micro-organism Interactions, INRAE Dijon) and Eric Vieren (UMR1347 Agroécologie) for
their help in growing the plants. We are particularly grateful to Anne-Lise Santoni (ECOLEG team of UMR1347 Agroécologie, now at the GISMO platform of the University of Burgundy) for N, C, and S analyses of seed tissues, and to Elise Sorin (UMR950 EVA) for her help in measuring sulfate concentration in plant tissues. We sincerely thank Jérôme Fromentin (UMR Agroécologie), Stéphane Mari, and Carine Alcon (IPSiM, Institut des Sciences des Plantes de Montpellier) for their assistance regarding the transient expression of PsSULTR4 in tobacco leaves. We would also like to thank in particular Nathalie Leborgne-Castel and Christophe Der (UMR1347 Agroécologie) for their helpful advice during the microscopic observations. We would like to thank Céline Masclaux-Daubresse (IJPB, Institut Jean-Pierre Bourgin) and Jean-Christophe Avice (UMR950 EVA), members of the PhD committee of Fanélie Bachelet, for fruitful discussions and helpful suggestions. We also thank Titouan Bonnot and Jonathan Kreplak for critical reading of the manuscript and helpful suggestions. For the purpose of open access, the authors have applied a CC-BY public copyright license to any Author Accepted Manuscript (AAM) version arising from this submission. This work was initiated in the framework of the FUI (French Inter-Ministerial Unique Funds)-SERAPIS project (No. F1209006) with grants from Timac Agro International and continued in the framework of the PEAVALUE project granted by the French National Research Agency (ANR-19-CE21-0008). Expression data during the kinetics of seed development were obtained using immature seeds collected in the framework of the REGULEG project (ANR-15-CE20-0001). The Ph.D. of Fanélie Bachelet was funded by UBFC (Université Bourgogne-Franche-Comté) through the ISITE-BFC Project (907.THESES.WP3 - Project PEASULTR4).

## CONFLICT OF INTEREST

The authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

## SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article.

**Figure S1.** Tonoplast localization of PsSULTR4 in mesophyll cells after transient expression of PsSULTR4-GFP in tobacco leaves.

**Figure S2.** Phylogenetic tree of SULTR4 protein sequences from legume species, with Arabidopsis as an outgroup.

**Figure S3.** Cartoon representation of the three-dimensional structure of AtSULTR4;1 predicted by AlphaFold.

**Figure S4.** Alignment of the PsSULTR4 sequence with putative SULTR4 proteins from representative species of each major lineage of land plants.

**Figure S5.** Cartoon representation of the predicted PsSULTR4 structure (AlphaFold) for each missense *sultr4* mutants.

**Figure S6.** Amounts of C and N in mature seeds of the *sultr4* mutants and wild-type plants grown under +S and -S conditions.

**Figure S7.** Relative expression of PsSULTR4 in leaves and developing seeds of pea plants 9 days after flowering.

**Table S1.** Taxonomy of the selected species used to generate the protein sequence alignments in Figure 2(B) and Figure S3, with their taxid from the NCBI database.

**Table S2.** Seed yield (g) at maturity for the five *sultr4* mutants and their corresponding wild-type plants grown under +S and -S conditions.

**Table S3.** Seed yield and yield components of pea plants grown under +S and -S conditions.

**Table S4.** C, N, and S contents in mature pea seeds and estimation of C, N, and S quantities in the seed compartment (all seeds) and per seed.

**Table S5.** S quantity (in mg) in each plant compartment at two stages (12 DAF and maturity) from individual pea plants (*sultr4* and wild types) grown under -S conditions.

**Table S6.** Phenotypic data (number of nodes and dry biomass for roots, vegetative, and reproductive parts) obtained at two stages (12 DAF and maturity) from pea plants (*sultr4* and wild types) grown under +S and -S conditions.

**Table S7.** Storage protein composition of mature seeds from *sultr4* and wild-type plants grown under +S and -S conditions as determined by FPLC.

**Table S8.** Primers used in this study.

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