Salicylic acid accumulation correlates with low anthocyanin production in Arabidopsis

Salicylic acid accumulation correlates with low anthocyanin production in Arabidopsis Matˇ ej Drs b,c,d , Oksana Iakovenko a , Jhonny Stalyn Orozco Hern´ andez a , Pavla Be´ ata Trhlínov´ a a , Vedrana Markovi´ c b,1 , Viktor ˇ Z´ arský b,d , Tamara Peˇ cenkov´ a b , Martin Janda a,* a Department of Experimental Plant Biology, Faculty of Science, University of South Bohemia in ˇ Cesk´ e Budˇ ejovice, Braniˇ sovsk´ a 1645/31a, ˇ Cesk´ e Budˇ ejovice, 370 05, Czech Republic b Laboratory of Cell Biology, Institute of Experimental Botany, Czech Academy of Sciences, Rozvojov´ a 263, Lysolaje, 165 02 Praha 6, Czech Republic c Imaging Facility, Institute of Experimental Botany, Czech Academy of Sciences, Rozvojov´ a 263, Lysolaje, 165 02 Praha 6, Czech Republic d Department of Experimental Plant Biology, Faculty of Science, Charles University, Viniˇ cn´ a 5, 128 44, Prague 2, Czech Republic ARTICLE INFO Keywords: AVI bodies Anthocyanins Salicylic acid mutants NahG Exocyst Autophagy ABSTRACT Anthocyanins, flavonoid pigments, are essential photoprotective agents and play a pivotal role in enhancing plant resilience to environmental stressors. It has been shown that anthocyanin production is inhibited when pattern-triggered immunity (PTI) is activated in Arabidopsis thaliana. An important component of PTI is the phytohormone salicylic acid (SA). Interestingly, exogenous treatment with SA has been shown to induce anthocyanin content in grape, apple, maize roots, rose callus, or Arabidopsis seedlings. In this study, we used several A. thaliana mutants with modulated SA content to decipher the role of endogenous SA in anthocyanin production in A. thaliana. We treated WT and mutants with anthocyanin-inducible conditions and measured anthocyanin content using spectroscopy. We showed that high endogenous SA accumulation correlates with low anthocyanin production. This was confirmed by the treatment of the A. thaliana seedlings with exogenous SA. Additionally, using microscopy in the 5gt mutant, which exhibits enhanced production of anthocyanin vesicular inclusions (AVIs) due to the inhibition of ligandin-dependent vacuolar import, we showed that high endogenous SA also correlates with lower AVI abundance. Comparative analysis of Arabidopsis WT and mutants used in this study indicates a possible inhibitory effect of SA accumulation on anthocyanin content under anthocyanininducible conditions (AICs). We suggest that under AICs, SA downstream signaling independent of NPR1 is responsible for lower anthocyanin accumulation. 1. Introduction Plants respond to various abiotic stresses, such as osmotic stress, UV light, and changes in day length, by inducing the synthesis of anthocyanins, which are water-soluble flavonoid pigments responsible for the characteristic red, blue, and purple colors of plant tissues. These pigments are synthesized via the phenylpropanoid pathway and represent glycosylated derivatives of anthocyanidins. Anthocyanidins are glycosylated at the 3-O and/or 5-O positions by the corresponding UDPglucose:flavonoid 3-O-glucosyltransferase (3 GT) or 5-O-glucosyltransferase (5 GT), respectively. Once synthesized, anthocyanins accumulate in the large central vacuole, where their oxidation is prevented. Their synthesis occurs at the cytoplasmic surface of the endoplasmic reticulum (ER), from where they are transported to the vacuole. Over the past years, several models have been proposed to explain anthocyanin transport to the vacuole (Li and Ahammed, 2023). The ligandin model suggests that anthocyanins are actively transported across the tonoplast by specific membrane importers, with the assistance of glutathione S-transferase (GST) enzymes (Poustka et al., 2007; Sun et al., 2012). The vesicular model proposes that anthocyanins, once imported into the ER lumen, are transported to the vacuole via vesicles (Gomez et al., 2011; Pourcel et al., 2010). Interestingly, the vesicular movement of anthocyanins—or anthocyanin vesicular inclusions (AVIs)—may also be regulated by microautophagy (Chanoca * Corresponding author. E-mail address: [email protected] (M. Janda). 1 Current address: Laboratoire Reproduction et Developpement des Plantes, Universit´ e de Lyon, ENS de Lyon, CNRS, INRA, Lyon, France. Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph https://doi.org/10.1016/j.jplph.2025.154604 Received 7 July 2025; Received in revised form 21 August 2025; Accepted 1 September 2025 Journal of Plant Physiology 314 (2025) 154604 Available online 2 September 2025 0176-1617/© 2025 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). et al., 2015; Pourcel et al., 2010). Anthocyanins have antioxidant properties, protecting plants against oxidative damage and high temperatures. Due to their coloration, anthocyanins also play an important role in attracting pollinators (Li and Ahammed, 2023). However, they are also induced by pathogen attack (Liu et al., 2020) and contribute to plant defense mechanisms by deterring herbivores and exhibiting antimicrobial properties (Lev-Yadun and Gould, 2008). On the other hand, it has been shown in Arabidopsis thaliana that activated pattern-triggered immunity (PTI), induced by treatment with the peptides elf18 and flg22, classical examples of microbe-associated molecular patterns (MAMPs), inhibits anthocyanin production triggered by high sucrose or UV-B irradiation (Schenke et al., 2011; Serrano et al., 2012; Zhou et al., 2017). Mitogen-Activated Protein Kinase Kinase 6 (MKK6) plays an important role in this inhibition (Wersch et al., 2018). A crucial integral component of PTI defence is also autophagy (Sertsuvalkul et al., 2022), and autophagy loss-of-function mutants accumulate high levels of salicylic acid, exhibit decreased levels of anthocyanins, and show enhanced spontaneous hypersensitive reaction (HR) (Masclaux-Daubresse et al., 2014). An integral component of PTI signaling is the phytohormone salicylic acid (SA) and its signaling pathway. SA concentration increases upon treatment with elf18 or flg22 (Tsuda et al., 2008). SA biosynthesis in plants occurs via two pathways, both starting from chorismate in the chloroplasts. The first is the isochorismate synthase (ICS)-dependent pathway, which is responsible for the majority of induced SA in A. thaliana during bacterial attack (Wildermuth et al., 2001). The second pathway depends on phenylalanine ammonia-lyase (PAL) (Dempsey et al., 2011). The ICS-dependent pathway appears to be more important Fig. 1. Expanding leaves accumulate more anthocyanins than well-developed leaves under the high sucrose and kinetin treatments, in a negative correlation with salicylic acid accumulation. A) Representative photos of WT A. thaliana plants before treatment with AICs and after treatments: with sucrose (72 h) and kinetin (168 h). Expanding (blue) and well-developed (red) leaves are highlighted. For better highlights of particular leaves we “normalized” the plant size to equal (bars, 10 mm). B) Anthocyanin content in expanding and well-developed leaves. C) Free salicylic acid (SA) and SA-glucoside (SAG) concentration in expanding and well-developed leaves. The analysis was performed before AIC treatment. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey test for (B) (p <0.01, n =7–8); for (C) unpaired t-test (p <0,001, n =16). Experiments were repeated 2–3 times. Bars represent 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) M. Drs et al. Journal of Plant Physiology 314 (2025) 154604 2 under stress conditions in Brassicaceae, whereas in other plant species, the PAL pathway plays a more prominent role (Ullah et al., 2023). The PAL-dependent biosynthetic pathway shares its initial step with the flavonoid biosynthesis pathway, and multiple mutations in PAL genes result in decreased levels of both SA and anthocyanins (Zhang et al., 2021). In this study, we focused on the role of the SA pathway in anthocyanin accumulation under abiotic stress conditions in Arabidopsis thaliana. 2. Materials and methods 2.1. Plant material We used Arabidopsis thaliana ecotype Columbia col-0 as a wild type (WT), additionally we used A. thaliana mutants with Col-0 genetic background which we used in previous studies: bon1-1, pi4kβ1/pi4kβ2, fah1/fah2, exo70B1-1, NahG, bon1-1/snc1-11, NahG/pi4kβ1/pi4kβ2 (Pluhaˇ rov´ a et al., 2019; Ortmannov´ a et al., 2022), tn2 (Zhao et al., 2015), tn2/exo70B1-1 (Zhao et al., 2015); previously published, but not by us 5gt (Chanoca et al., 2015) and which we generated in this study: 5gt/exo70B1-1 and 5gt/exo70B1-1/tn2. Plants used for analyses were grown in three distinct growing conditions. Either A. thaliana plants were grown in hydroponia using ¼ Hoagland solution, and after 4–5 weeks, treated by anthocyanininducing conditions (AICs; particularly treated either with sucrose or kinetin). The temperature was set to 20 ±2 ◦C, light intensity was 130–150 μ mol m −2 .s −1 , and photoperiod was 10 h/14 h (light/dark) (these growing conditions were used for results in Fig. 1). Alternatively, plants were cultivated in Jiffy palettes for 5 weeks under the short day conditions (8/14; light intensity 100 μ mol m −2 .s −1 ; 70 % humidity); additionally, the seedlings were cultivated in liquid ½ MS in 6-wells plates (Deltalab, Spain) for 5 days under the long days conditions (16/ 8; light intensity 100 μ mol m −2 .s −1 ). 2.2. Anthocyanin-inducing conditions For plants grown in hydroponia, the AIC 7 % sucrose or 50 μ M kinetin (diluted in 5M KOH) was added to fresh fresh-prepared ¼ Hoagland solution. A new ¼ Hoagland solution was used as a mock. For the kinetin experiment, the mock was ¼ Hoagland solution with KOH (50 μ l/L 5M KOH). The induction of anthocyanins accumulation for Jiffy tablets cultivated plants was performed by the switch from the short day condition were transferred into a chamber with a long day (16/8; light intensity 100 μ mol m −2 .s −1 ; 70 % humidity) and cultivated for 14 days. The accumulation of anthocyanins in seedlings was triggered by adding of fresh or ½ MS with 7 % sucrose into cultivation wells with seedlings for 48 h treatment (these growing conditions were used for the results in Figs. 2 and 3). 2.3. Salicylic acid treatment At IEB 5-day-old seedlings grown in liquid ½ MS were treated in cultivation wells with ½ MS containing 7 % sucrose with or without different concentrations of SA (40, 100, 200 and 400 μ M) for 48 h. Treatments with 200 and 400 μ M SA are shown in Fig. 3, while the lower concentrations (40 and 100 μ M) are presented in Fig. S2. 2.4. Anthocyanin analysis 2.4.1. Spectrofotometry For anthocyanins measurement from A. thaliana grown in hydroponia fully developed adult leaves were used. Detached leaves were homogenized in the extraction solution (methanol with 1 % HCl), vortexed and centrifuged at 20 000 RCF for 20 min. Absorbance was measured using a spectrophotometer (Analytik Jena Specord 210 Plus), and anthocyanin content was calculated by the formula ((λ 528 -λ 657 )/4)/ FW (Zou et al., 2017). Anthocyanin isolation from whole seedlings was done with the same method. 2.4.2. Microscopy Cotyledons or mature leaves were mounted onto microscopic slides and observed using the Olympus (BX-51 with Olympus DP-74 camera) microscope with a real-time z-stack stitching plugin (Olympus-cellSens software). AVIs were analyzed using FIJI (Schindelin et al., 2012) with a labkit plugin to segment AVIs from the background. 2.5. Salicylic acid analysis with SA-biosensor Acinetobacter sp. ADPWH_lux For SA measurement of fully developed and expanding leaves and pi4kβ1/pi4kβ2-related mutants was used method based on bacterial biosensor described in (DeFraia et al., 2008; Lu et al., 2018). Briefly, 4–5 week old plants were used for SA measurements. For set of mutants shown in Fig. S1 (pi4kβ1/pi4kβ2, NahG; NahG/pi4kβ1/pi4kβ2) we used fully developed leaves; for WT we analyzed fully developed leaves and expanding leaves. Acinetobacter sp. ADPWH_lux OD600 =0.4 was used for measurements. Plant material was collected in liquid nitrogen and grind with mortae and pestle, 50–100 mg was put into 1.5 mL eppy. NaAC buffer (250 μ L/100 mg) was added to a sample. Kept on ice. Vortex thoroughly. After centrifugation (12 000 RCF, 10 ◦C, 10 min) pellet was discarded and supernatant used for incubation with Acinetobacter. For SAG measurement 50 μ L of supernatant was used for treatment with β-glucosidase (2 U per sample; 90 min for 37 ◦C). Standard curve was created using salicylic acid (Sigma-Aldrich; ref. nr. 105910). The assay was performed in white 96-well plate and luminescence was measured using Tecan Spark microplate reader (TECAN). 3. Results and discussion 3.1. High endogenous salicylic acid reduces anthocyanin accumulation Since previous reports on anthocyanin accumulation, conducted on various plant species and tissues under biotic stress conditions, have yielded conflicting results, we aimed to analyze the effect of both endogenous and exogenous SA on anthocyanin accumulation in Arabidopsis thaliana using distinct experimental approaches. We treated plants cultivated in hydroponia with 7 % sucrose or with 50 μ M kinetin, typical anthocyanin-inducing conditions (AICs) (Figs. 1 and 2). During the establishment of AIC conditions in our laboratory, we observed that the anthocyanin production is dependent on leaf age. Anthocyanin accumulation was faster in expanding leaves than in welldeveloped leaves (Fig. 1A and B). It was shown in Arabidopsis, tomato and tobacco that SA concentration is higher in older leaves (Han et al., 2025; Zhang et al., 2013). We measured SA content in expanding and well-developed leaves and we observed that older leaves have higher SA concentration (Fig. 1C). Thus, anthocyanin accumulation under AICs seems to negatively correlate with SA level in the leaves. To verify this anthocyanin-SA negative correlation under established AICs we tested fully developed leaves from collection of Arabidopsis mutants with distinct SA content changes: fah1/fah2; bon1-1; pi4kβ1/ pi4kβ2 and exo70B1 with high SA content (Pluhaˇ rov´ a et al., 2019; Ortmannov´ a et al., 2022) and corresponding double mutants bon1-1/snc1-11; NahG/pi4kβ1/pikβ2 and exo70B1/tn2 with normalized low SA content (Pluhaˇ rov´ a et al., 2019; Zhao et al., 2015); for comparison we used WT and NahG with lowered SA content due to SA degradation (ˇ Saˇ sek et al., 2014). NahG is a transgenic plant expressing bacterial salicylate hydroxylase metabolizing SA to katechol (Delaney et al., 1994). Our results demonstrated that high endogenous SA correlates with low anthocyanin accumulation in AICs (Fig. 2). In all double mutants with normalized WT SA level (bon1-1/snc1-11, exo70B1/tn2 M. Drs et al. Journal of Plant Physiology 314 (2025) 154604 3 Fig. 2. High endogenous salicylic acid inhibits anthocyanin production. Roots of 4-5-week-old Arabidopsis plants grown hydroponically were treated with (A) 7 % sucrose for 72 h and (B) 50 μ M kinetin for 168 h. C) Representative images of Arabidopsis plants after sucrose and kinetin treatment. Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey test (p <0.05, n =5–8 independent samples). Experiments were repeated 2–3 times for each genotype and treatment. Red color indicates mutants with high SA concentration; green color indicates mutant with low SA concentration. Bars represent 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) M. Drs et al. Journal of Plant Physiology 314 (2025) 154604 4 and NahG/pi4kβ11/pi4kβ2), we observed restored anthocyanin production (Fig. 2). This reinforces the phenomenon that SA and anthocyanin accumulation negatively correlate (Masclaux-Daubresse et al., 2014). Results showing strong production of anthocyanins in NahG/pi4kβ1/pikβ2 and certainly in NahG alone (Fig. 2), demonstrate that not SA biosynthesis flux but SA accumulation or SA downstream signaling is responsible for lower anthocyanin accumulation. The master regulator of SA signaling is the NPR1 protein (Janda and Ruelland, 2015). Kulich et al. (2013) showed that crossing of npr1 knock-out mutant with the exo70B1 mutant did not restore anthocyanin accumulation in exo70B1 under AICs (Kulich et al., 2013). Additionally, Zhao et al. (2015) showed that double mutant npr1/exo70B1 has even higher content of SA than the exo70B1 single mutant (Zhao et al., 2015). Taken together, we suggest that SA alone is not an inhibitor of anthocyanin production, but SA signaling (e.g. changes in metabolic flux caused by SA), including the NPR1-independent pathway, is responsible for the inhibition of anthocyanin accumulation under AICs. However, the detailed mechanism of that inhibition remains to be elucidated. 3.2. Exogenous application of SA decreases vacuolar anthocyanin accumulation, including anthocyanin vacuolar inclusion bodies (AVIs) Mutant plants from our variable SA content collection provided information about the inhibitory effect of accumulation of endogenous SA Fig. 3. Exogenous SA inhibits anthocyanin production and alters vacuolar inclusion bodies. A) Plot showing the analysis of anthocyanin content in seedlings of WT and 5gt mutant under anthocyanin-inducing conditions (AIC; 7 % sucrose), combined with salicylic acid (SA) treatment at two concentrations (200 and 400 μ M). B) Diagram illustrating the effect of the 5gt mutation on anthocyanin transport. C) RGB images of cotyledon leaves under the same treatments as in (A); scale bar =50 μ m (. Red arrows indicate AVIs (Anthocyanin Vacuolar Inclusions); Analysis of AVI properties: abundance (D), size (E), and circularity (F). Statistical analysis was performed using one-way ANOVA followed by a post hoc Tukey test (p <0.001). n =at least 16 independent leaves per genotype (D, E, F); 12 seedlings per genotype per treatment with 3 technical replicates for (A). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) M. Drs et al. Journal of Plant Physiology 314 (2025) 154604 5 on the ability to produce and accumulate anthocyanins (Fig. 2). However, it was shown that exogenous treatment with SA triggers increase of anthocyanin content in Arabidopsis (Liu et al., 2020), grape berries and cells (Khalili et al., 2022; Obinata et al., 2003; Oraei et al., 2019; Yue et al., 2023), in pomegranate (García-Pastor et al., 2020), in pepper plants (Mahdavian et al., 2008), in Rosa hybrida calli (Ram et al., 2013), in maize roots (Jain and Srivastava, 1984), in combination with elevated CO 2, also in ginger (Ghasemzadeh et al., 2012), in combination with sucrose in Pistacia chinensis leaves (Song et al., 2020), and SA alleviated the inhibitory effect of ethylene on anthocyanin production in Brassica napus (Tirani et al., 2013). Since the kinetin and sucrose treatments had the same AIC effect, we proceeded further on with less costly and more stable sucrose, and we combined it with exogenously added SA. We observed, surprisingly a direct inhibitory effect of exogenous SA on the amount of anthocyanins under AIC in a concentration-dependent manner in A. thaliana seedlings (Fig. 3A). Along with the spectroscopy, we also used a second approach for the quantification of anthocyanins vacuolar accumulation analysing anthocyanin vacuolar inclusions (AVIs) bodies in 5gt mutant as a genetic background. 5gt mutants cannot deliver soluble anthocyanins to the vacuole via ligandin-dependent importer pathway but use an autophagy-related vesicular pathway, leading to excessive accumulation of insoluble AVIs that can be quantified inside the vacuole microscopically (Pourcel et al., 2010) (Fig. 3B). AVIs of SA-treated plants were less abundant, smaller and more circular than AVIs observed in control plants treated just with AIC. It supports our previous observations with absorbance measurements of anthocyanin content. Surprisingly, the abundance of AVIs does not decrease significantly between 200 μ M SA and 400 μ M SA, indicating a possible saturation effect of 200 μ M SA. On the other hand, the area and circularity of AVIs are extensively affected by increased concentration of SA. AVIs in SA treated 5gt plants tend to be smaller and more rounded than AVIs observed in control AIC-only conditions. Results from experiments using treatment with exogenous SA are in accordance with our data from mutants with high endogenous SA (Fig. 2). Our observations differ from what has been described in other plant species after treatment with exogenous SA, where SA had a positive effect on anthocyanin production, as mentioned above in this chapter. Liu et al. (2020) showed that anthocyanin accumulation caused by infection with Penicillium corylophilum correlates with increased SA content in Arabidopsis. Additionally, they demonstrated that treatment with exogenous SA induces anthocyanin production, and that this effect is dependent on NPR1 (Liu et al., 2020). Thus, it appears that SA has a dual role in regulating anthocyanin accumulation. The effect of SA and its downstream signaling depends on environmental conditions. If plants are grown under AICs, high SA levels may inhibit anthocyanin accumulation. Conversely, if the plant is not under apparent stress, elevated SA may trigger a stress response that leads to anthocyanin accumulation. We speculate that the observed correlation between low anthocyanin accumulation and high SA levels could result from an ongoing spontaneous hypersensitive response (HR) in SA-overaccumulating mutants. This is a common phenomenon in such mutants, including autophagy mutants, and may also involve potential metabolic feedback. 3.3. SA-triggered reduction of anthocyanin vacuolar accumulation occurs independently from anthocyanin vesicular transport to the vacuole Both pi4kβ1/pi4kβ2 and exo70B1 mutants exhibit defects in endomembrane vesicular trafficking (Kang et al., 2011; Kulich et al., 2013). However, expression of NahG in the pi4kβ1/pi4kβ2 background fully alleviates the inhibitory effect on anthocyanin accumulation by downregulating SA content. While SA hyperaccumulation is reduced by the tn2 mutation in the exo70B1 background (Zhao et al., 2015), this only partially relieves the anthocyanin accumulation defect (Fig. 2). It suggests a potentially more prominent role for EXO70B1 in overall anthocyanin accumulation. To verify whether vesicle-dependent trafficking of anthocyanins into the vacuole is affected by elevated SA levels in an EXO70B1-dependent manner, we used a collection of 5gt, 5gt/exo70B1, and 5gt/tn2/exo70B1 mutants. As highlighted above, in the 5gt mutant background, the major carrier-dependent pathway for anthocyanin delivery to the vacuole is disrupted, and transport relies on the vesicular/autophagy machinery. Indeed, AVIs in exo70B1/5gt adult plants, which have high SA content, were significantly less abundant than in the 5gt single mutant, but normalized in the exo70B1/tn2/5gt triple mutant (Fig. 4). Thus, SA downregulation in the exo70B1/tn2 double mutant background restores the ability to accumulate anthocyanins via vesicular transport, suggesting that SA acts upstream to inhibit anthocyanin accumulation independently of EXO70B1-mediated vesicular uptake into the vacuole. Overall, our results imply that high endogenous SA, characteristic of exo70B1 and several other mutants used in this study, has an inhibitory effect on overall anthocyanin synthesis and accumulation, including the 5GT/ligandin-dependent carrier import pathway. These observations indicate that SA accumulation correlates with reduced anthocyanin levels through currently unknown mechanisms that affect the overall availability or synthesis of anthocyanins, independent of their vacuolar delivery pathways. 4. Conclusion We showed that high endogenous SA (and possibly its downstream NPR1-independent signaling, as suggested by (Kulich et al., 2013)) competes with or inhibits anthocyanin accumulation independently of both the ligandin-dependent and vesicular import pathways to the vacuole. These observations contribute to our understanding of how activated pattern-triggered immunity may inhibit anthocyanin production. CRediT authorship contribution statement Matˇ ej Drs: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Oksana Iakovenko: Writing – review & editing, Visualization, Methodology, Investigation. Jhonny Stalyn Orozco Hern´ andez: Writing – review & editing, Methodology, Investigation. Pavla Be´ ata Trhlínov´ a: Methodology, Investigation. Vedrana Markovi´ c: Writing – review & editing, Methodology, Investigation. Viktor ˇ Z´ arský: Writing – review & editing, Supervision, Conceptualization. Tamara Peˇ cenkov´ a: Writing – review & editing, Supervision, Data curation, Conceptualization. Martin Janda: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Data curation, Conceptualization. Usage of generative AI During the preparation of this work the author(s) used copilot and Grammarly in order to improve the English. After using this tool/service, the author(s) reviewed and edited the content as needed and take (s) full responsibility for the content of the publication. Funding This work was supported by Ministry of Education, Youth and Sports (MEYS) (from the EU Operational Programme), the nr. CZ.02.2.69/0.0/ 0.0/18_053/0016975 (MJ), by MEYS mobility project nr. 8J23FR034 (MJ, OI), by MEYS project TowArds Next GENeration Crops, reg. no. CZ.02.01.01/00/22_008/0004581 of the ERDF Programme Johannes Amos Comenius (TP, VZ), by Student Grant Agency at Faculty of Science, University of South Bohemia (PBT), Charles University Grant Agency (GAUK) project 415322 (MD). Ministry of Education, Youth and Sports “National Infrastructure for Biological and Medical Imaging (Czech-BioImaging – LM2023050)" (MD). M. Drs et al. Journal of Plant Physiology 314 (2025) 154604 6 Declaration of competing interest The authors declare no conflict of interest. Acknowledgements We thank Petra Fialov´ a for her excellent support. We also thank Martin Potocký for support and Jana ˇ S ť ovíˇ ckov´ a for technical assistance during the research. We thank to the imaging facility at Institute of Experimental Botany AS CR v.v.i. 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