Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid

Plant Science 344 (2024) 112087 Available online 9 April 2024 0168-9452/© 2024 Elsevier B.V. All rights reserved. Arabidopsis seeds altered in the circadian clock protein TOC1 are characterized by higher level of linolenic acid Salim Makni a , S´ ebastien Acket a , St´ ephanie Guenin b , Sana Afensiss a , Adeline Guellier a , Raquel Martins-Noguerol c , Antonio J. Moreno-Perez d , Brigitte Thomasset a , Enrique Martinez-Force e , Laurent Gutierrez b , Eric Ruelland a , Adrian Troncoso-Ponce a , * a Unit´ e de G´ enie Enzymatique Et Cellulaire (GEC), CNRS UMR 7025, Universit´ e de Technologie de Compi` egne, Compi` egne 60203, France b Centre R´ egional de Ressources en Biologie Mol´ eculaire (CRRBM), Universit´ e Picardie Jules Verne, 33 rue Saint-Leu, Amiens 80039, France c Department of Plant Biology and Ecology, Faculty of Biology, University of Seville, Sevilla, Spain d Department of Microbiology, Faculty of Biology, University of Seville, Seville, Spain e Instituto de la Grasa-CSIC, Building 46, UPO Campus, Seville, Spain ARTICLE INFO Keywords: Circadian clock Lipid biosynthesis FA composition Seeds Pseudo-response regulators TOC1 ABSTRACT The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds. 1. INTRODUCTION Lipids are a major and essential component of cells. In plants, we can distinguish several lipid classes, including glycerolipids (GL), sphingolipids, waxes, cutins and suberins (Li-Beisson et al., 2013). GL are either composed of three fatty acids (FA) esterified to a glycerol backbone, and thus known as triacylglycerol (TAG), or consist of two FA and a polar head group bound to the glycerol skeleton. The latter are referred to as phospholipids (phosphatidylcholine: PC, phosphatidylserine: PS, phosphatidylethanolamine: PE, phosphatidylglycerol: PG and phosphatidylinositol: PI), glycolipids (monogalactosyldiacylglycerol: MGDG and digalactosyldiacylglycerol: DGDG) and sulfolipids (sulfoquinovosyldiacylglycerol: SQDG) depending on the nature of the polar head group (Li-Beisson et al., 2013). In addition to their structural functions in membranes, phospholipids, glycolipids and sulfolipids are involved in signal transduction, photosynthesis and protection against biotic and abiotic stresses (Kobayashi et al., 2016). On the other hand, TAG provides energy and carbon to plants during early germination before the establishment of photosynthesis (Graham, 2008). The biosynthesis of GL is preceded by the de novo FA synthesis, mostly palmitic (16:0), palmitoleic (16:1), stearic (18:0) and oleic (18:1) acids (Focks and Benning, 1998; Li-Beisson et al., 2013). Once synthesized, these FA are exported to the endoplasmic reticulum (ER) to be elongated into very long chain FA (VLCFA), including eicosanoic (20:0), eicosenoic (20:1), docosanoic (22:0), docosenoic (22:1) and tetracosanoic (24:0) acids (Li-Beisson et al., 2013). Thereafter, the incorporation of two FA, followed by the addition of either a polar head group or a third FA to the glycerol backbones, results in the synthesis of GL by the prokaryotic or eukaryotic pathway (Li-Beisson et al., 2013). The prokaryotic pathway assembles GL within plastids, while the eukaryotic pathway within the ER (Li-Beisson et al., 2013). Even though in developing seeds de novo FA synthesis produces saturated and monounsaturated FA, polyunsaturated GL are formed within the ER. The enzymes fatty acid desaturase 2 and 3 (FAD2 and FAD3) are responsible for producing 18:2-PC and 18:3-PC. The subsequent transfer of these FA from PC to new glycerol backbones allows for the formation of polyunsaturated TAG (Bates et al., 2009, 2012; Menard et al., 2018). In plants, physiological processes associated with seed maturation, * Corresponding author. E-mail address: [email protected] (A. Troncoso-Ponce). Contents lists available at ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci https://doi.org/10.1016/j.plantsci.2024.112087 Received 26 November 2023; Received in revised form 4 March 2024; Accepted 8 April 2024 Plant Science 344 (2024) 112087 2 including FA synthesis and accumulation of TAG, are tightly regulated through the action of several transcription factors, namely LEAFY COTYLEDON 1 (LEC1), LEAFY COTYLEDON 2 (LEC2), ABSCISSIC ACID INSENSITIVE 3 (ABI3), FUSCA 3 (FUS3), WRINKLED 1 (WRI1), and LEC1-like (L1L) (Baud et al., 2009; Baud and Lepiniec, 2009; Kumar et al., 2020; Ruuska et al., 2002). During seed maturation, LEC1, LEC2, ABI3 and FUS3 positively regulate the expression of WRI1, which promotes FA synthesis (Focks and Benning, 1998; Baud et al., 2007). The transcription factor L1L increases the expression of the gene basic LEUCINE ZIPPER TRANSCRIPTION FACTOR 67 (bZIP67), a FAD3 regulator (Yamamoto et al., 2009; Mendes et al., 2013). Another regulatory network, the circadian clock, contributes to the control of physiological processes during the entire life of the plant. The circadian clock enables plants to anticipate rhythmic fluctuations imposed by the Earth’s rotation, such as changes in light and temperature, allowing the plant to adapt its physiology to varying external conditions. The circadian clock regulates the stomatal aperture, flowering and carbohydrate synthesis (Dodd et al., 2005; Graf et al., 2010; Pokhilko et al., 2013). Moreover, Hudson (2010) and Nakamura et al. (2014) highlighted the potential involvement of the circadian clock in the regulation of lipid metabolism in soybean seeds and Arabidopsis leaves, respectively. In the study conducted by Hudson (2010), microarray data obtained from soybean developing seeds showed a cyclic expression profile under continuous light for genes with predicted functions in lipid metabolism, including FAD2. Similarly, Nakamura et al. (2014) reconstructed an oscillatory expression pattern for genes involved in the synthesis of several GL under continuous light (http://diurnal.mocklerlab.org/) (Mockler et al., 2007). The plant circadian clock consists of multiple interlocking feedback loops (Adams et al., 2015). The central loop involves two morning-expressed MYB-like transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and a single evening-expressed pseudo-response regulator protein TIMING OF CAB EXPRESSION 1 (TOC1, also known as PRR1) (Alabadi et al., 2001). The expression of CCA1/LHY is repressed by TOC1 during the night, while the expression of TOC1 is reduced by CCA1/LHY during the day (Adams et al., 2015). Besides this central loop, other clock components, such as PRR9, PRR7 and PRR5, regulate the expression of CCA1/LHY and TOC1 (Adams et al., 2015; Huang and Nusinow, 2016). CCA1/LHY and TOC1 play pivotal roles in modulating numerous plant physiological processes throughout the day and night. In Arabidopsis, CCA1 and LHY are involved in the regulation of FA and GL metabolism within seeds, both clock components participate in the synthesis of phosphatidic acid (PA) and enhance TAG accumulation (Kim et al. 2019, 2023). Whereas, Gendron et al. (2012) showed that TOC1 serves as a general transcriptional regulator, influencing the expression of over 2500 genes in Arabidopsis, this regulation includes the tricarboxylic acid cycle-related gene FUMARASE2. TOC1 suppresses FUMARASE2, thereby decreasing fumarate accumulation. This mechanism helps maintaining energy homeostasis and controls plant metabolism during the night (Cervela-Cardona et al. 2021). TOC1 binds to two distinct DNA sequences: the TOC1 morning element (T1ME: TGTG) and, in complex with phytochrome interacting factor (PIF), the G-box (CACGTG) (Gendron et al., 2012). While the T1ME sequence is specific to the promoters of CCA1 and LHY, the G-box sequence is more widespread, appearing in the promoters of various genes, including those encoding the desaturases FAD2 (Kim et al., 2006) and FAD3 (Mendes et al., 2013), as well as the fatty acid elongase 1 (FAE1) (Du et al., 2011). Although the transcription factors LEC1, LEC2, ABI3, FUS3, WRI1, and L1L have been identified as orchestrating FA synthesis during Arabidopsis seed development (Baud et al., 2009; Baud and Lepiniec, 2009; Kumar et al., 2020; Ruuska et al., 2002), the importance of CCA1 and LHY during this process has recently been highlighted (Kim et al., 2019, 2023). In cca1lhy seeds, a significant decrease in oil content was observed. Conversely, LHY-overexpressing seeds exhibited an increase in their oil content. LHY binds and activates the promoter of the gene encoding for the β-ketoacyl-ACP synthase III (KASIII), the enzyme responsible of the first condensation during FA synthesis (Kim et al., 2023). However, to the best of our knowledge, the role of the circadian clock in regulating FA metabolism during seed maturation remains incompletely understood. Therefore, the aim of the current study was to gain insight into the connections between seed FA metabolism and TOC1, which, in conjunction with CCA1/LHY, forms the core of the Arabidopsis circadian clock. 2. MATERIALS AND METHODS 2.1. Plant materials and growth conditions Arabidopsis thaliana seeds used in this manuscript include the previously described toc1–1, toc1–2 (Strayer et al., 2000), TOC1-OE (Makino et al., 2002), prr5–1, prr7–3, and prr9–1 (Michael et al., 2003). We thank Dr. Manuel Rodriguez-Concepcion, Dr. Takafumi Yamashino and Dr. Elena Monte for sharing seeds. Arabidopsis seeds were sterilized by adding 1 mL of 70% ethanol containing 0.01% SDS for 3 min and 1 mL of 20% of the commercial bleach solution for 20 min (Lindsey et al., 2017) and were then germinated on Murashige and Skoog medium. The resulting seedlings were grown for 1 month in 12 h light/12 h dark condition, 70% humidity and 22◦C. Seedlings were transferred to autoclaved soil and cultivated in a growth chamber maintained at 22◦C and 50% humidity under light cycles of 12 h light/12 h dark condition. 2.2. Fatty acid analyses Fatty acid methyl ester (FAME) were generated using the whole seed transmethylation protocol (Li-Beisson et al., 2013). This protocol was also adapted for developing leaves and seedlings. In short, 1 mL of methanol containing 5% H 2 SO 4 , 50 µg/mL of internal standard (17:0 for seeds or 15:0 for seedlings and leaves) and 50 µg/µL of butylated hydroxytoluene (BHT) was added to 50 mature seeds, 5 mg of seedlings or 5 mg of leaves. Next, 300 µL toluene was added before vortexing for 30 seconds. The derivatization of FA into FAME was then performed by heating samples at 85◦C for 90 min. After cooling for 5 min at room temperature, 1.5 mL of 0.9% NaCl and 1 mL of heptane were added. Samples were centrifuged for 2 min at 4000 rpm. The heptane phases were evaporated under nitrogen stream and resuspended into 50 µL of heptane. FAMEs were then analyzed by GC-FID equipped with a BPX70 column as described by Tao et al., (2021). 2.3. GL extraction and characterization GL were extracted from mature seeds as described by Folch et al., (1951) with some modifications. The extraction solution consisted of chloroform/methanol (2:1) containing 50 µg/mL of BHT and 1 µM of internal standard (SPLASH Lipidomix, Avanti Polar Lipids, inc.). The addition of 1 mL of the extraction solution and 400 µL of water to 20 mg of mature seeds was followed by the homogenization of the samples (Precellys; 5500 rpm, 3 ×90 sec). After cooling on ice for 15 min, samples were centrifuged for 5 min at 4◦C and 13,000 rpm. The phase containing GL were transferred in a 2 mL vial before evaporating under nitrogen stream. The evaporated lipid extract was resuspended in 400 µL of chloroform. Lipid extract was 4-fold diluted and stored at −20◦C until the analysis by High-Performance Liquid Chromatography coupled by a Quadrupole Time-Of-Flight (HPLC-QTOF). For seedlings, the extraction of GL was performed as described by Tsugawa et al. (2020). The extraction mix consisted of methyl tert-butyl ether (MTBE)/methanol (3:1) containing 1 µM of internal standard (SPLASH Lipidomix, Avanti Polar Lipids, inc.) and 50 µg/mL of BHT. 1 mL of the extraction mix and 400 µL of water were added to 100 mg of seedlings. Samples were vortexed and homogenized by a Precellys (5500 rpm, 3 ×90 sec). After cooling on ice for 15 min, samples were centrifuged at 5500 rpm for 10 min at 4◦C and the phases containing GL were transferred to a new tube. A second extraction of GL was performed by adding 750 µL of S. Makni et al. Plant Science 344 (2024) 112087 3 MTBE to the seedling. Extractions were combined, evaporated under nitrogen stream, resuspended in 60 µL of isopropanol and centrifuged at 12,000 rpm for 10 min at 4◦C. Supernatant was stored at −20◦C until the analysis by HPLC-QTOF. GL extracted from mature seeds and seedlings were analyzed by HPLC-QTOF as described by Tsugawa et al. (2020) and Tao et al. (2021). To summarize, the HPLC system from Agilent Technologies (HPLC 1290) was equipped by the ACQUITY UPLC CSH C18 column (100 ×2.1 mm; 1.7 μ m) and ACQUITY UPLC CSH C18 VanGuard pre-column (5 ×2.1 mm; 1.7 μ m). The column and pre-column were maintained at 65◦C. The separation was done at 0.6 mL/min by using the mobile phase 60:40 (v/v) acetonitrile/water (solvent A) and 90:10 (v/v) isopropanol/acetonitrile (solvent B) implemented of 10 mM of ammonium acetate for negative mode or 10 mM ammonium formate and 0.1% formic acid in positive mode, as previously described (Tao et al., 2021). The files generated by Agilent (*. d) were converted to the *.mzML format using MSConvert (Chambers et al., 2012). Data were processed and lipids were annotated by using Ms-Dial 4, as previously described by Tao et al. (2021). The peak height was used as the intensity measure for each annotated lipid in the mass spectra. The normalization of data was performed using an appropriate internal standard. Once peak tables were exported into.csv format, the mol% of each GL molecular species was determined. Lipidomic data were imported into MetaboAnalyst 5.0 platform (http://www.metaboan alyst.ca/) (Pang et al., 2021). 2.4. RNA analyses RNA extraction was performed on developing siliques at 12 DAF (days after flowering) and developing seeds at 16 DAF. Plants grown in soil were tagged when flowers emerged and siliques were harvested at 12 and 16 DAF. The former were ground in liquid nitrogen and from the latter, developing seeds were excised using a scalpel and forceps before being ground in liquid nitrogen. In both cases, biological material was collected 1 hour before the beginning of the dark period. Grinded biological materials were transferred in 2 mL of ice-cold extraction buffer constituted of 2% of β-mercaptoethanol freshly added to 8 M of LiCl. Samples were homogenized, stored overnight at 4◦C, vortexed for a second time and centrifuged at 13,000 rpm for 10 seconds at 4◦C. Once transferred to new 2 mL tubes, RNA-containing supernatants were centrifuged at 13,000 rpm for 30 min at 4◦C. Supernatants were discarded and RNA-containing pellets were resuspended by vortexing in 750 µL of ice-cold 75% ethanol. Samples were centrifuged at 13,000 rpm for 5 min at 4◦C. Supernatants were removed and tubes were then left open for 5–10 min in order to evaporate residual ethanol. The pellet was resuspended in 600 µL of solubilization buffer consisting of 2% of β-mercaptoethanol freshly added to 0.5% SDS, 100 mM NaCl, 25 mM EDTA and 10 mM Tris-HCl (pH 7.6). RNA was washed from other components, including polyphenol, by adding 600 µL of phenol/chloroform/isoamyl alcohol mix (25/24/1, v/v/v) to the solubilization buffer, vortexing for 30 seconds and centrifuging at 13,000 rpm for 5 min at room temperature. The upper phase containing RNA, corresponding to the solubilization buffer fraction, was transferred to 1.5 mL tubes. RNA was washed from polyphenol two other times by repeating the steps indicated above. About 400 µL of RNA was recovered after the last washing. Twenty-seven µL of 4 M NaCl and 800 µL of ice-cold 100% ethanol were added to 400 µL of supernatants. RNA precipitation took place at −80◦C for 30 min. Samples were then centrifuged at 13,000 rpm for 15 min at 4◦C, supernatants were discarded and the RNA-containing pellet was washed three times by adding 500 µL of ice-cold 75% ethanol. Each washing step was followed by the centrifugation of sample at 13,000 rpm for 5 min at 4◦C. The final step of the RNA extraction consisted in the resuspension of the pellet in 30 µL of sterile water. Genomic DNA was then eliminated using a recombinant and modified form of the DNase I. The protocol is described in the TURBO DNAse-free™ kit user manual (Fisher Scientific, Invitrogen). For the reverse transcription, 100 pmol of Oligo(dT) 18 were added to 0.1–5 µg of RNA and the RNA/Oligo (dT) 18 mix was then filled with milli-Q water up to 12.5 µL. To these 12.5 µL were introduced 4 µL of the 5X Reaction Buffer, 20 U of the Ribolock RNase inhibitor, 20 nmol of each dNTP and 200 U of the RevertAid Reverse Transcriptase, reaching a total volume of 20 µL. The 20 µL mix was then incubated at 42◦C for 1 h and 70 ◦C for 10 min allowing the synthesis of cDNA from the RNA template. Once synthesized, cDNA samples were diluted to 1/20. Transcript levels were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using a LightCycler 480 system (Roche), including reference gene validation, EF1 α , as previously described in Gutierrez et al. (2012). The sequences of primers were indicated in Table S1. 2.5. Statistical analysis Statistical analyses were performed by Student’s t-test using the Microsoft Excel. Statistical details including the number of replicates for each figure and table are indicated in their corresponding legends. In addition to Student’s t-test, further statistical analyses, including Principal Components Analyses (PCA), volcano plots and ANOVA-based heatmap, were performed using MetaboAnalyst 5.0 platform. 3. RESULTS 3.1. TOC1 influences Arabidopsis seed FA composition To investigate the involvement of the circadian clock PRR family proteins, specifically PRR9, PRR7, PRR5, and TOC1, in the regulation of FA metabolism in Arabidopsis seeds, we analyzed the FA composition of seeds harvested from prr9–1, prr7–3, prr5–1, toc1–1, toc1–2 and TOC1- OE plants. Non-statistical differences were found in terms of size or weight between mutant seeds and their corresponding WT. The prr5–1, prr7–3 and prr9–1 plants, generated from Col-0, resulted from the introduction of a single T-DNA sequence in the first exon of PRR5 and in the second exon of PRR7 and PRR9 (Michael et al., 2003). In the toc1–1, generated from C24 ecotype, a missense mutation in the exon 6 produced TOC1 proteins with reduced DNA-binding properties (Strayer et al., 2000; Gendron et al., 2012). The toc1–2 mutation, obtained from Col-0 background, was characterized by a modification of the last guanidine of the exon 1 into adenosine, thus leading to the production of TOC1 transcript in which the 13 nucleotides following the sequence of the first exon were not correctly spliced (Strayer et al., 2000). In the TOC1-overexpressing line (TOC1-OE), the CaMV-35S promoter drives the overexpression of TOC1 in the Col-0 ecotype. (Makino et al., 2002). The circadian clock rhythm of the TOC1-OE plants exposed to continuous light was estimated to 30 h against 24 h in its WT counterpart (Makino et al., 2002), while the period of the circadian clock rhythm was about 20 h in toc1–1 and toc1–2 plants cultivated in the same light conditions (Strayer et al., 2000). Additionally, these TOC1-OE plants were, in contrast to toc1–2, insensitive to a 3-day high-temperature treatment. This phenotype is the consequence of TOC1-OE blocking the expression of PIF4-mediated temperature-responsive genes (Zhu et al., 2016). We chose this particular TOC1-overexpressing line for our study. Our results showed that toc1–1 and toc1–2 exhibited the most significant differences in terms of FA composition compared to their respective WT seeds (Fig. 1). Volcano plots showed that both toc1 mutants shared a similar FA profile characterized by higher content of 18:0, 18:3, 20:0, and 22:1, and lower content of 18:2 (Fig. S1). In the case of toc1–1, the increase corresponded to 35% for 18:0, 12% for 18:3, 43% for 20:0, and 11% for 22:1 and the decrease to 18% for 18:2. Similarly, in the case of toc1–2, an increase of 43%, 13%, 37% and 5% for 18:0, 18:3, 20:0 and 22:1, respectively, and a reduction of 16% for 18:2 was observed. The FA profiles of prr9–1, prr7–3, and prr5–1 seeds exhibited minor modifications (Fig. S1 and S2). The FA composition of seeds harvested from TOC1-OE plants was also analyzed, revealing a significant decrease in 22:1 and an increase in 18:0 and 20:0 (Fig. 1 and S1). According to the volcano plot, in TOC1-OE seeds the composition of 18:2 S. Makni et al. Plant Science 344 (2024) 112087 4 Fig. 1. FA composition in TOC1-altered seeds. a, b) Contents of individual FA expressed as mol%; c, d) Contents of total FA expressed as μ g/mg. Values are mean ±SD with individual data points from six biological and three technical repetitions. Statistical significance was evaluated by a bilateral Student’s t test using unpaired samples (P <0.05: *, P <0.01: ** and P <0.001: ***). Fig. 2. Principal Component Analysis of GL in TOC1-altered seeds. PCA score plot showing the similarity relations among Arabidopsis seeds harvested from controls and TOC1-altered plants. Samples from control lines are shown as red circles, those from toc1 and TOC1-OE as green and orange circles respectively. S. Makni et al. Plant Science 344 (2024) 112087 5 and 18:3 was not changed, in contrast to both toc1 mutants (Fig. S1). The accumulation of total FA decreased (11%) in TOC1-OE seeds and increased in toc1–1 and toc1–2 (9% and 11%, respectively) (Fig. 1). Then, the FA compositions of 1-month-old seedlings and 5-week-old leaves were examined. As shown by Fung-Uceda et al. (2018), toc1–2 plants were associated with a dwarf phenotype due to a stronger alteration of their development (Fig. S3). Therefore, the comparative FA analysis of seedlings and leaves was carried out only in toc1–1 plants. In seedlings, a reduction in 18:2 (15%) and an increase in 16:3 (18%) were observed, while a decrease in 18:2 (17%) was shown in leaves (Fig. S1 and S4). However, differences in the composition of FA observed in both tissues remain lower than 18%, while reaching up to 43% in seeds. 3.2. Lipidomic differences reflect modifications in FA composition in toc1 and TOC1-OE seeds GL composition in TOC1-altered and WT seeds were investigated. High-performance liquid chromatography coupled by a mass spectrometer (HPLC-MS) was used due to the high diversity of GL species found in plants (Li et al., 2014). In WT seeds a total of 118 species corresponding to 61 TAG, 19 PC, 18 DAG, 11 PE, 4 DGDG, 4 lyso-PC, and 1 MGDG were annotated in positive mode (Table S2). No GL were detected in negative mode. Thereafter, unsupervised PCA was employed to visualize differences in GL profiles obtained from TOC1-altered seeds (Fig. 2). In both analyses, a clear separation between toc1 mutants and their respective control counterparts underscored their differences in GL composition. In contrast, these differences were lower between TOC1-OE and Col-0 as shown by the limited separation obtained in the PCA analysis. A hierarchical clustering was used to highlight these modifications, and an ANOVA-based heatmap of the 45 GL species displaying the most significant differences was generated (Fig. 3). Volcano plots identified 41 and 45 significantly different species in toc1–1 compared to C24 and toc1–2 compared to Col-0, respectively (Fig. S5). Thirty of these species were common to both toc1 mutants (Table S3). Furthermore, a comparison between the TAG profiles of toc1 seeds revealed that toc1–1 and toc1–2 shared an increase in TAG containing 18:0, 18:3, 20:0 and 22:1, accompanied by a decrease in those with 18:2 moieties (Fig. 4 and Table S3). The GL composition in TOC1-OE showed only 27 GL species significantly modified when compared to Col-0 (Fig. S5); among these species, those containing 22:1 were decreased (Table S3). In order to investigate the repercussions of toc1 mutation on GL metabolism in vegetative tissues, GL profiles of seedlings from toc1–1 and C24 plants were obtained and compared. The analysis of GL from 1-month-old seedlings was performed in both positive and negative modes. In positive mode 6 DAG, 4 DGDG, 7 MGDG, 17 PC, 10 PE, 1 PG and 4 TAG species were annotated, while 11 MGDG, 3 PA, 8 PC, 9 PE, 9 PG, 3 PI and 8 SQDG were detected in negative mode. Volcano plot (Fig. S5) and Table S4 showed 17 and 23 GL species significantly modified between toc1–1 and C24 seedlings in positive and negative modes, respectively, while significant differences were observed for 41 GL species between toc1–1 and C24 seeds (Fig. S5). 3.3. Expression of FA-related genes in toc1-1 and TOC1-OE To gain a deeper understanding of the participation of TOC1 in the regulation of FA metabolism during seed maturation, we compared the expression, by RT-qPCR, of selected FA-related genes in toc1–1 and TOC1-OE. These genes were selected based on the altered FA profile previously obtained in toc1 seeds. The analysis was conducted on 16 DAF seeds and 12 DAF siliques. In both cases, samples were harvested 1 h before the beginning of the dark period (Zeitgeber 11; ZT11), which corresponds to the peak level of TOC1 expression (Huang and Nusinow, Fig. 3. Hierarchical clustering and heatmap of GL in TOC1-altered seeds. Heatmap representing the most significantly different GL between TOC1-altered and WT seeds. The heatmap corresponds to lipidomic data generated from C24 (red), toc1-1 (green), Col-0 (dark blue), toc1-2 (light blue) and TOC1-OE (pink). S. Makni et al. Plant Science 344 (2024) 112087 6 2016). TOC1 expression was not modified in toc1–1 seeds, while increased in toc1–1 and TOC1-OE siliques (Fig. 5). The expression levels of genes encoding enzymes involved in FA metabolism, including KASIII (AT1G62640), FAD2 (AT3G12120), FAD3 (AT2G29980), and FAE1 (AT4G34520), were then analyzed. Additionally, the expressions of bZIP67 (AT3G44460) and WRI1 (AT3G54320) transcription factors were also examined. The expression levels of all these genes were found to be increased in toc1–1 seeds, while in toc1–1 siliques, only FAD3, FAE1, and WRI1 showed upregulation. No significant differences were observed in the expression of these genes in TOC1-OE siliques (Fig. 5). 4. DISCUSSION The presence of an active circadian clock in Arabidopsis seeds, although necessary for the response to dormancy-breaking signals (Penfield and Hall, 2009), has been sparsely studied in comparison with its role during germination (Arana et al., 2017; Yang et al., 2021). One of the few studies focusing on connecting the circadian clock and the physiology of developing seeds has recently highlighted the particpation of CCA1/LHY in regulating FA synthesis (Kim et al., 2023). In the present study, among all the mutants analyzed (toc1–1, toc1–2, prr5–1, prr7–3, prr9–1, and TOC1-OE), toc1–1 and toc1–2 displayed the most significant modifications in terms of seed FA composition. Indeed, in contrast to PRR5, PRR7 and PRR9, TOC1 together with CCA1/LHY Fig. 4. TAG composition in TOC1-altered seeds. Contents of individual TAG expressed as mol%. Data are average values ±SD from three biological and three technical repetitions. Statistical significance was evaluated by a bilateral Student’s t test using unpaired samples (P <0.05: *, P <0.01: ** and P <0.001: ***). S. Makni et al. Plant Science 344 (2024) 112087 7 regulate the expression of several genes related to plant metabolism, including genes involved in the tricarboxylic acid cycle and lipid metabolism (Hsiao et al., 2014; Cervela-Cardona et al., 2021; Kim et al., 2019; 2023). Our data showed a reduction in total FA content in TOC1-OE seeds and an increase in toc1–1 and toc1–2. In contrast, oil seed content was decreased in lhycca1 and increased in LHY/CCA1-OEs (Kim et al., 2023). These opposite lipid phenotypes point to the reciprocal regulation between CCA1/LHY and TOC1. These results suggest the participation of TOC1, together with CCA1/LHY, in the regulation of FA metabolism during seed development. Despite the deeper alteration of TOC1 function in toc1–2, characterized by the production of a truncated and non-functional protein, as opposed to toc1–1 in which only DNA binding properties are reduced (Strayer et al., 2000; Gendron et al., 2012), similarities in FA and GL seed profiles were obtained. Therefore, confirming the importance of the transcription factor activity of TOC1 during FA synthesis. The seed FA composition of toc1–1 and toc1–2 showed an increase in 18:3 together with a decrease in 18:2, whereas lhycca1 seeds had a decrease in 18:2 but 18:3 was not modified (Kim Fig. 5. Expression of FA-related genes in TOC1-altered plants. Expression of selected FA-related genes in 16 DAF seeds and 12 DAF siliques harvested at ZT11. Gene expression was normalized to the expression of the reference gene EF1 α . Data are average values ±SD from four biological and two technical repetitions. Statistical significance was evaluated by a bilateral Student’s t test using unpaired samples (P <0.05: *, P <0.01: ** and P <0.001: ***). S. Makni et al. Plant Science 344 (2024) 112087 8 et al., 2023). In the present study, GL analyses were obtained as complementary approaches to the FA determination. As observed for FA, results showed similarities in TAG profiles in toc1–1 and toc1–2, TAG containing 18:0, 18:3, 20:0, and 22:1 were increased, while TAG with 18:2 moieties were decreased. In addition, TAG containing 22:1 were decreased in TOC1-OE seeds. Therefore, the differences in TAG composition reflected the altered FA profiles observed in TOC1-altered seeds. Taking into account the FA profiles observed in toc1 seeds, our results pointed to the involvement of TOC1 in the regulation of FAD3, the main seed desaturase producing 18:3 (Browse et al., 1993). Given that FAD3 expression is positively modulated by bZIP67 (Mendes et al., 2013), TOC1 potentially regulates the expression of FAD3 and bZIP67. The decrease in 18:2 content in toc1 seeds suggested that TOC1 controls the expression of FAD2, highly expressed in developing seeds (Okuley et al., 1994). Moreover, the increase in VLCFA indicates the potential role of TOC1 in the regulation of FAE1, one of the genes involved in their synthesis (James et al., 1995). In addition to these enzymes, the respective increase and decrease in the FA seed content in toc1 and TOC1-OE suggested that TOC1 could also participate in the regulation of KASIII and WRI1 expression. The enzyme KASIII catalyzes the initial condensation reaction in the sequential addition of two-carbon units leading to FA synthesis. Recently, Kim et al. (2023) demonstrated that LHY/CCA1 bind to the promoter of KASIII, enhancing its expression and FA synthesis in Arabidopsis seeds. WRI1 is a transcription factor involved in the positive regulation of the late glycolysis and FA synthesis during seed maturation (Focks and Benning, 1998; Cernac and Benning, 2004). To confirm all these hypotheses, KASIII, FAD2, FAD3, bZIP67, FAE1 and WRI1 expressions were evaluated by RT-qPCR. The expression pattern of FA-related genes in Arabidopsis developing seeds showed elevated transcript levels from 6 to 16 DAF, peaking at 12 DAF (Baud et al., 2009; Baud and Lepiniec, 2009; Ruuska et al., 2002), and the active accumulation of FA from 10 to 16 DAF (Baud et al., 2002). The expression of the circadian clock components is usually expressed in Zeitgeber time (ZT), a unit of time based on a precise moment of a 12 h light/12 h dark cycle with ZT0 and ZT12 corresponding to dawn and dusk, respectively. In plants, the expression of TOC1 increases from ZT0 to ZT12, reaching a high level at ZT11 (Huang and Nusinow, 2016). Altogether, the expression of FA-related genes at ZT11 was assessed in TOC1-altered plants at two different developmental time points and in two different tissues. Initially, the study focused on 16 DAF immature toc1–1 seeds. Later, the analysis extended to include the expression of the same genes in 12 DAF toc1–1 and TOC1-OE siliques. It is worth noting that although the latter tissue consists of seeds surrounded by maternal silique walls, it has also been used to investigate the expression of seed-related genes (James et al., 1995; Ruuska et al., 2002; Baud et al., 2007; 2009). Concerning 16 DAF toc1–1 seeds, our results showed that TOC1 expression was not modified at transcriptional level. Indeed, toc1–1 mutation only affects the ability of TOC1 to interact with its target DNA sequence (Strayer et al., 2000; Gendron et al., 2012). However, the expression of KASIII, FAD2, FAD3, bZIP67, WRI1 and FAE1 was increased. The increase in FAD3, bZIP67, FAE1, KASIII and WRI1 expressions explained the higher content of 18:3, VLCFA and total FA observed in toc1–1 seeds. The expression of KASIII displayed opposite changes in toc1–1 and lhycca1 seeds, being upregulated in the former and downregulated in the latter (Kim et al., 2023), in accordance with oil contents found in both mutants and the reciprocal regulation between CCA1/LHY and TOC1 (Alabadi et al., 2001). Interestingly, although FAD2 expression was increased, 18:2 content was decreased in these seeds. This disagreement between enzyme expression and product accumulation reflexes the complexity of interactions within the FA synthesis pathway. As above-mentioned, the transcriptional analysis was extended to 12 DAF toc1–1 siliques. In these tissues, in contrast with the pattern obtained in developing seeds, the expression of TOC1 was increased. The upregulation of TOC1 observed in siliques is likely due to the existence of a positive transcriptional feedback in the maternal silique walls, aiming at compensating the lack of efficient TOC1 protein. As previously observed in developing mutant seeds, the expression of FAD3, FAE1 and WRI1 was significantly higher in toc1–1 siliques. However, the expression of KASIII, FAD2 and bZIP67 was not modified. This discrepancy could be consequence of the potential dilution of the mRNA extracted from developing seeds with those extracted from the maternal silique walls. Finally, the expression of these genes were studied in 12 DAF TOC1-OE siliques. As expected, the expression of TOC1 was increased in TOC1-OE, while the expression of lipid-related genes remained unchanged. In conclusion, the upregulation of lipid-related genes together with the modifications found in the lipid composition of TOC1-altered seeds, prove the participation of TOC1 in the regulation of seed FA metabolism. These results, combined with the ones recently published by Kim et al. (2023), highlight the importance of the transcription factors belonging to the circadian central loop, CCA1/LHY and TOC1, in the regulation of lipid metabolism during seed maturation in Arabidopsis plants. CRediT authorship contribution statement Enrique Martinez-Force: Methodology, Investigation, Formal analysis, Data curation. Laurent Gutierrez: Investigation, Formal analysis, Data curation, Methodology. Eric Ruelland: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. Manuel Adrian Troncoso Ponce: Writing – original draft, Supervision, Resources, Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration. Salim Makni: Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Writing – original draft. Adeline Guellier: Data curation, Investigation. S´ ebastien Acket: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – original draft. St´ ephanie Guenin: Methodology, Investigation, Formal analysis, Data curation. Sana Afensiss: Formal analysis, Data curation. Raquel Martins-Noguerol: Methodology, Investigation, Formal analysis. Antonio Moreno-Perez: Methodology, Investigation, Formal analysis, Data curation. Brigitte Thomasset: Resources, Investigation, Funding acquisition, Formal analysis. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgement We acknowledge Dr. Manuel Rodriguez-Concepcion (Institute of Molecular & Cellular Biology of Plants, Spain), Dr. Takafumi Yamashino (Nagoya University, Japan) and Dr. Elena Monte (Center for Research in Agricultural Genomics, Spain) who kindly provided the Arabidopsis mutant seeds. We thank Andrew Huss for its careful and critical reading of the manuscript. Finally, we would like to thank the “Minist` ere français de lʼEnseignement sup´ erieur, de la Recherche et de lʼInnovation”, the Hauts-de-France region and the European Regional Development Fund (ERDF) for the funding of this work. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.plantsci.2024.112087. S. Makni et al. Plant Science 344 (2024) 112087 9 References S. Adams, I. Manfield, P. Stockley, I.A. 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