# Increase of histone acetylation by suberoylanilide hydroxamic acid enhances microspore reprogramming and expression of somatic embryogenesis transcription factors in Brassica napus

Yolanda Pérez-Pérez, Eduardo Berenguer, Elena Carneros, Pilar S Testillano

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Yolanda Pérez-Pérez, Eduardo Berenguer, Elena Carneros, Pilar S Testillano. Increase of histone acetylation by suberoylanilide hydroxamic acid enhances microspore reprogramming and expression of somatic embryogenesis transcription factors in Brassica napus. Plant Science, 2025, 351, pp.112318. 10.1016/j.plantsci.2024.112318. hal-05101516

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# Increase of histone acetylation by suberoylanilide hydroxamic acid enhances microspore reprogramming and expression of somatic embryogenesis transcription factors in *Brassica napus*

Yolanda Pérez-Pérez<sup>a,1</sup>, Eduardo Berenguer<sup>a,b,1</sup>, Elena Carneros<sup>a</sup>, Pilar S. Testillano<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

- Microspore embryogenesis

- Pollen development

- Epigenetics

- Histone post-translational modifications

- SAHA

## ABSTRACT

*In vivo*, microspores in the anthers follow the gametophytic development pathway, culminating in the formation of pollen grains. Conversely, *in vitro*, under stress treatments, microspores can be reprogrammed into totipotent cells, initiating an embryogenic pathway that produces haploid and double-haploid embryos, which are important biotechnological tools in plant breeding. There is growing evidence that epigenetic reprogramming occurs during microspore embryogenesis through DNA methylation, but less is known about the role of histone modifications. This study investigates the dynamics of histone acetylation during the two microspore developmental pathways, microspore embryogenesis and pollen development, in *Brassica napus*. We analyzed histone H3 and H4 acetylation levels, histone acetyltransferase (HAT) activity and expression of *HAC5* acetyltransferase using immunofluorescence, enzymatic activity assays, ELISA-like tests, and qPCR. Results showed a decrease in global histone acetylation levels during pollen maturation, correlated with reduced HAT activity and down-regulation of the *BnHAC5-like* gene. In contrast, stress-induced microspore reprogramming led to increased histone acetylation levels, enhanced HAT activity, and upregulation of *BnHAC5-like*. Treatment with sub-eroylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor widely used in animal research but barely applied to plants, enhanced microspore embryogenesis initiation and proembryo formation, while increased histone acetylation levels. SAHA-treated proembryos showed higher expression than control of key embryogenesis transcription factors *BnFUS3*, *BnAGL15*, and *BnLEC2*, suggesting that histone hyperacetylation facilitates transcriptional activation of essential genes for somatic embryogenesis initiation. These findings provided new insights into the epigenetic regulation of this process and revealed new opportunities with histone epigenetic regulator inhibitors, to improve microspore embryogenesis induction for crop improvement.

## 1. Introduction

Cellular plasticity in plants, as demonstrated by their capacity to regenerate organs and embryos through *in vitro* cultures, is a hallmark of the plant kingdom, extensively utilized in biotechnology for the propagation, conservation, and improvement of species significant to agriculture, forestry, and industry (Germaná and Lambardi, 2016). The method of *in vitro* somatic embryogenesis, initiated across a diverse range of cell types such as haploid microspores, exemplifies the remarkable adaptability inherent to plant cells. Under specific inductive conditions, the microspores can attain complete embryogenic competence, leading to the formation of whole embryos, a process that has

generated great interest in both research and biotechnological applications (Fehér, 2015).

In their natural state within the anther, microspores undergo a strictly regulated gametophytic development, culminating in the production of pollen grains capable of fertilization. However, when isolated and subjected to specific stress treatments in an *in vitro* environment, the microspores acquire the potential to become cells capable of generating haploid and double-haploid embryos, a radical change from their original cellular destiny. This process, called stress-induced microspore embryogenesis, has become a crucial method in plant breeding. It enables the accelerated creation of double-haploid plants, prized for their uniform genetic variability and the ability to fix desired genetic traits in

<footnote><sup>a</sup> Pollen Biotechnology of Crop Plants Group, Margarita Salas Center of Biological Research, CIB-CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain</footnote>

<footnote><sup>b</sup> Plant Reproduction and Development Laboratory, ENS de Lyon, CNRS, INRAE, UCBL, Lyon F-69342, France</footnote>

<footnote>* Corresponding author.
E-mail address: testillano@cib.csic.es (P.S. Testillano).</footnote>

<footnote n="1">¹ Both authors contributed equally.</footnote>
just one generation (Horstman et al., 2017; Maluszynski et al., 2003).

Despite recent advancements providing insights into some aspects of somatic embryogenesis, a lack of knowledge persists concerning the complex chain of events that orchestrate the transformation of somatic cells towards embryo formation. Factors such as stress response, hormonal activity, and epigenetic mechanisms are considered key components in achieving cell reprogramming and totipotency, and triggering embryogenesis in microspores (Testillano, 2019), as well as in the embryogenic development of other cell types (Díaz-Sala, 2019; Fehér, 2015).

Depending on the plant species, microspore embryogenesis can be conducted by subjecting microspores to diverse stress treatments, such as thermal shocks, nutrient deprivation, or alteration of osmotic conditions (Islam and Tuteja, 2012; Shariatpanahi et al., 2006). Specifically, in *Brassica napus*, a model system for inducing cellular reprogramming, microspores are reprogrammed towards embryogenesis through the application of a mild heat stress treatment at 32°C (Prem et al., 2012).

Epigenetic mechanisms, particularly histone modifications, have been described as significant regulatory factors in plant response and tolerance to certain types of stresses, as well as during specific developmental programs (Hu et al., 2019). The DNA within chromatin is structured into nucleosome sequences, each of which is constituted by an octamer consisting of two copies of each histone protein - H2A, H2B, H3, and H4 - with DNA wrapped around this complex (Luger et al., 1997). The N-terminal tails of histones contain numerous post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. These modifications directly affect the physical association with DNA or serve as a docking or blocking accessory for other additional proteins. Therefore, the positioning of nucleosomes, the post-translational modifications of histones, the replacement of histones by variants, and the methylation of DNA contribute to the three-dimensional folding of DNA and influence the activation or repression of transcription at specific loci (Friedrich et al., 2019).

Histone acetylation and methylation are among the most extensively studied and well-characterized epigenetic marks. In previous studies, we investigated the dynamics of histone H3 methylation in lysine 9 during microspore embryogenesis (Berenguer et al., 2017). In the present study, we have focused on examining the role of another relevant epigenetic mark: histone acetylation. Histone acetylation involves the addition of an acetyl group, provided by the molecule of acetyl coenzyme A (CoA), to the ε-amino group at the N-terminal end of the lysine residue. Acetylation of lysines neutralizes the positive charges between the amino group of histone proteins and the negative charges of DNA. These modifications provide a more open chromatin conformation state, facilitating the binding of regulatory proteins or transcription factors (TFs) to DNA, and the consequent activation of transcription (Hu et al., 2019).

The levels of histone acetylation are determined by the balance between the activity of the enzymes histone acetyltransferases (HATs) and histone deacetylases (HDACs or HDAs). Numerous studies have revealed that the interaction between HATs and HDACs plays a key role in epigenetic regulation during certain developmental processes and in response to environmental conditions (Kumar et al., 2021).

In the present study, we have examined the dynamics of global histone H3 and H4 acetylation and the activity and expression of acetyl transferase enzymes during *in vivo* gametophytic development in *Brassica napus*, comparing these profiles with those observed following *in vitro* microspore reprogramming and embryogenesis initiation. Furthermore, we have conducted functional assays with suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylases widely used in animal research but barely applied to plants, to elucidate its impact on the embryogenic induction of the microspore.

The results obtained consistently showed an opposite pattern of global histone acetylation during both developmental programs of the microspore, pollen development and microspore embryogenesis, while

demonstrated that SAHA treatment increased acetylation levels, enhanced microspore reprogramming and embryogenesis initiation, and promoted the expression of key somatic embryogenesis-related transcription factors.

## 2. MATERIAL AND METHODS

### 2.1. Plant material and microspore embryogenesis in vitro cultures

Donor rapeseed plants (*Brassica napus* L. cv. Topas, line DH407) were cultivated under controlled conditions as previously described (Pérez-Pérez et al., 2023). Microspores and pollen grains developed *in vivo* were isolated from anthers to analyse the gametophytic pathway. *In vitro* cultures of isolated vacuolated microspores, the most responsive stage to embryogenesis induction, were performed as previously described, employing a stress treatment at 32°C (Prem et al., 2012).

### 2.2. Sample processing for microscopy analyses

The samples were fixed in 4 % paraformaldehyde (PF) in a phosphate-buffered saline (PBS) solution at a pH of 7.3 and kept at 4°C overnight. Early-stage culture samples containing isolated microspores and small multicellular pro-embryos were initially pre-embedded in gelatin, and processed for resin embedding. Larger multicellular embryos, which included globular and torpedo forms, were processed for either resin embedding or cryostat sectioning. Resin sections were stained with toluidine blue for structural microscopic analyses and cryostat sections were used for immunofluorescence assays.

For the resin embedding process, after fixation, and gelatin pre-embedding (for microspores and proembryos), the samples were rinsed with PBS, dehydrated using a graded acetone series, and embedded in Technovit 8100 resin (Kulzer) at 4°C. Semithin sections of resin, with a thickness of 2 µm, were prepared using an Ultracut E Reichert ultramicrotome and mounted on standard microscope slides for staining with 1 % (w/v) toluidine blue. Alternatively, they were stored at 4°C on multiwell slides coated with APTES (Sigma-Aldrich) for subsequent immunofluorescence analysis, following the protocol outlined by Pérez-Pérez et al. (2023).

For cryostat sectioning, the fixed samples were rinsed with PBS and gradually infiltrated with sucrose solutions of increasing concentrations (0.1, 0.5, 1, 1.5, and 2 M for one hour each, and 2.3 M overnight at 4°C) to cryoprotect the samples. They were then embedded in Tissue-Tek optimal cutting temperature (OCT) compound and frozen on dry ice for sectioning with a Leica CM 1950 cryostat. Sections with a thickness of 20–30 µm were collected on glass slides, rinsed with water to remove the Tissue-Tek OCT, transferred to a drop of water on slides coated with 3-aminopropyl-triethoxy-silane (APTES), air-dried, and stored at –20°C until ready for immunofluorescence analysis.

### 2.3. Immunofluorescence and confocal microscopy analyses

Immunofluorescence assays were carried out to localize histone H3 acetylated (H3Ac) and histone H4 acetylated (H4Ac) across different *in vivo* and *in vitro* developmental stages. Cryostat sections required permeabilization, after thawing the sections at room temperature, they were dehydrated and rehydrated in a methanol series (30, 50, 70, 90, 100, 90, 70, 50, 30%; 5 min each) and PBS and subjected to enzymatic digestion of cell walls for additional permeabilization with 2.5 % pectinase, 2.5 % cellulose, and 2.5 % pectoliase in PBS for 45 min. Semithin resin sections and cryostat sections on multiwell APTES-coated slides were washed with PBS for 5 min, blocked by 5 % (w/v) BSA (Sigma-Aldrich) in PBS for 10 min, then washed again with PBS and incubated for 1 hour with the corresponding primary antibody (H3Ac polyclonal antibody Millipore 06–599 or H4Ac polyclonal antibody Millipore 06–598) diluted 1:10 in 1 % (w/v) BSA in PBS. Following PBS washes, all sections were incubated with Alexa Fluor 488-labelled anti-rabbit IgG
antibody (Molecular Probes), diluted 1:25 in 1% (w/v) BSA in PBS, for 45 minutes in darkness. Finally, sections were counterstained with 1 mg/mL DAPI (Fluka) for 10 min, mounted in Mowiol® 4–88 (Sigma-Aldrich) and analyzed using a confocal microscope (Leica TCS-SP5 AOBS). To ensure a precise comparison of immunofluorescence signal intensities across developmental stages, confocal microscopy images were acquired using the same laser excitation and sample emission settings for each tested antibody. Control experiments were performed by replacing the primary antibody with PBS.

## 2.4. Quantification of global H4 acetylation

Histone extraction and global acetylation of histone H4 were measured using the EpiQuick™ Global Histone H4 Acetylation Kit (EpiGentek) according to the manufacturer's instruction. For protein extraction, a minimum of 50 mg of each sample was homogenized in a mortar with liquid nitrogen. Histone protein concentration was determined using the Bradford method and adjusted to 300 ng mL⁻¹ for the enzymatic assay. The acetylated histone H4 was detected using a specific antibody and a HRP-conjugated secondary antibody-color development system. The color intensity was quantified by measuring absorbance employing a Multiskan™ Sky Microplate UV/VIS reader (ThermoFisher Scientific) at 450 nm. The ratio of acetylated H4 in each sample was calculated relative to the positive and negative controls provided in the kit.

## 2.5. HAT activity detection

The HAT activity assay was performed using the EpiQuick™ HAT Activity/Inhibition Assay Kit (EpiGentek), in accordance with the guidelines provided by the manufacturer. Protein extracts from each developmental stage were obtained and adjusted as previously described, using a final concentration of 5 µg µL⁻¹ for the enzymatic reaction. During the assay, histone substrate underwent acetylation by HATs, and the resulting acetylated histones were detected using a specific antibody in an ELISA-like reaction. The quantity of acetylated histone was measured colorimetrically showing a direct correlation with HAT enzymatic activity. The color intensity was assessed by measuring absorbance using a microplate reader at 450 nm. To ensure precise calculations, the slope of the standard curve generated from the HAT Assay Standard, tested at various concentrations (0.1, 0.5, 1, 5 and 10 ng), was determined to calculate the HAT activity of each sample.

## 2.6. Expression analyses by qPCR

Gene expression levels were quantified using quantitative polymerase chain reaction (qPCR). RNA extraction from both anther and culture samples was conducted using the RNeasy® Plant Mini kit (Qiagen), followed by purification with NucleoSpin® RNA Plant (Ma-cherey-Nagel, Düsen, Germany), following the manufacturer's guidelines. The quality, concentration and purity of the extracted RNA were assessed with a NanoDrop ND-1000 spectrophotometer (ThermoFisher Scientific). Subsequently, cDNAs were synthesized from 1 µg of total RNA, using Superscript™ II reverse transcriptase (Invitrogen). Quantitative real-time PCR was conducted utilizing the SsoAdvancedTM Universal

SYBR Green Supermix on the iQTM5 Real-Time PCR Detection System (Biorad). The information about the oligonucleotides employed for the expression analysis of the BnHAC5-like, BnFUS3, BnAGL15 and BnLEC2 genes is presented in Table 1. As internal reference gene, HELICASE (BnHEL) was used, as it has been validated in previous studies (Berenguer et al., 2021, 2020; Carneros et al., 2024) as a reliable reference for this biological system. Its suitability for qPCR assays is due to its stable expression across various developmental stages in B. napus microspore culture samples, confirmed through independent amplification experiments (Berenguer et al., 2021, Supplementary Table S1). For each qPCR reaction, 25 ng of cDNA from the corresponding sample and primers at 500 nM concentration were used. Thermocycle settings were carried out as described by Pérez-Pérez et al., (2023). Data analysis was performed using the LightCycler®96 software (v.1.1.0.1320) (Roche Diagnostics International Ltd.), applying the Livak calculation method (Livak and Schmittgen, 2001). Transcript levels were expressed as mean values of relative expression standardized against the vacuolated microspore stage for both pollen development and microspore embryogenesis, using isolated vacuolated microspores in this latter case. Within each developmental program, variations among stages were evaluated using one-way ANOVA followed by Tukey's multiple comparison test, with statistical significance established at $p \le 0.05$.

## 2.7. In vitro treatment with the histone deacetylase inhibitor SAHA

In vitro treatments using the inhibitor suberoylanilide hydroxamic acid (SAHA, Epigentek) were assayed on microspore cultures. SAHA was added to the microspore liquid culture media by using a stock solution of 10 mM SAHA in dimethyl sulfoxide (DMSO). Appropriate volumes of the SAHA stock solution were added to the medium of culture plates, at the beginning of microspores culture, to obtain 1, 2.5 or 5 µM working concentrations). Additionally, mock plates without the drug served as experimental controls. The effect of SAHA on microspore induction efficiency was evaluated by quantifying the percentage of proembryos (the earliest morphological evidence of embryogenesis initiation) produced in 4-day cultures, from three independent experiments and at least three culture plates examined in each experiment for each condition. Mean percentages of proembryos were determined from randomly obtained micrographs using an inverted microscope Leica DMI6000B and a stereomicroscope Leica MZ16F.

## 2.8. Data analysis

Statistical differences among different stages within each developmental program and among different drugs conditions were evaluated using one-way ANOVA followed by Tukey's multiple comparison test, with statistical significance established at $p \le 0.05$. A minimum of three biological and three technical replicates were included in each statistical analysis.

<table><caption>**Table 1**

Primer sequences used for RT-qPCR assays.</caption><thead><tr><th rowspan="2">Gene</th><th rowspan="2">Accession number</th><th rowspan="2">Product size</th><th colspan="2">Primer sequences (5' → 3')</th></tr><tr><th>Forward</th><th>Reverse</th></tr></thead><tbody><tr><td>AtHEL</td><td>AT1G58050</td><td>61 bp</td><td>CCATTCTACTTTTTGGCGGCT</td><td>TCAATGGTAACTGATCCACTCTGATG</td></tr><tr><td>BnHAC5-like</td><td>Bra039359</td><td>176 bp</td><td>AGCCGAGTACACTTGCCTA</td><td>GCCTAGCCCTCTCCTGTCTT</td></tr><tr><td>BnFUS3</td><td>DY012343.1</td><td>152 bp</td><td>GTCACCGCCACAGCCACTAC</td><td>CTCGACTGAGCCAAAACCCTCG</td></tr><tr><td>BnAGL15</td><td>DQ418546.1</td><td>150 bp</td><td>TGTGCTTGAAGATGGGTCGTGG</td><td>CAATGACGGCAACCTCAGCGT</td></tr><tr><td>BnLEC2</td><td>DY021430.1</td><td>154 bp</td><td>TCAACTTCCATCACATCACCA</td><td>GCTCCTCGGAAATCTAAACGA</td></tr></tbody></table>
## 3. RESULTS

### 3.1. Changes in nuclear distribution pattern of acetylated histones H3 and H4 during microspore embryogenesis and pollen development

As a first step, the cellular organization at main stages of pollen development under study was characterized. Within the anthers, following male meiosis, the tetrad of microspores was produced surrounded by a temporary callose wall. Subsequently, the microspores were released from the tetrad and progressed to become vacuolated microspores, characterized by a round or elongated nucleus and a large vacuole which cause the nucleus to be displaced towards the cell periphery (Fig. 1A). Following asymmetric division, the vacuolated microspore developed into a bicellular pollen grain containing a smaller generative cell within the cytoplasm of the larger vegetative cell (Fig. 1B). Upon mitosis of the generative cell, the tricellular pollen was formed, consisting of a vegetative cell and two sperm cells (Fig. 1C).

In vitro, under certain conditions, the inherent developmental trajectory of the microspore can be altered. In *Brassica napus*, when subjected to a heat stress treatment (32°C), isolated vacuolated microspores, the most responsive stage to embryogenesis, can be reprogrammed, transforming into totipotent cells that proceed along an embryogenic pathway as previously described (Prem et al., 2012). The responsive microspores changed their developmental program after the stress treatment, initiating an embryogenic process that led to the formation of multicellular structures known as proembryos, still surrounded by the microspore wall, the exine, (Fig. 1D), formed after 4 days from the beginning of culture. These structures are considered the first sign of embryogenesis initiation. Over the subsequent days of culture,

microspore embryos developed and followed a zygotic embryogenesis-like pathway typical of dicotyledonous plant. After 10 days, globular embryos were observed (Fig. 1E), which developed into heart-shaped embryos (Fig. 1F) and subsequently into torpedo embryos (Fig. 1G). Finally, mature cotyledonary embryos are differentiated after 30 days in culture (Fig. 1H).

To analyse the changes in nuclear localization pattern of acetylated histones, immunofluorescence assays were performed in key stages of both developmental pathways. The assays were performed using antibodies against acetylated H3 and H4 during pollen development and microspore embryogenesis. The immunofluorescence intensities and labelling patterns obtained were similar with the two antibodies, in all the developmental stages analysed (Figs. 2 and 3).

In the gametophytic development, results exhibited an intense fluorescence signal of acetylated histone H3 (Fig. 2A-A'') and acetylated H4 in the nucleus of the vacuolated microspore. However, the fluorescence intensity for the two acetylated histones decreased in more advanced stages, in the bicellular and tricellular pollen, and vegetative, generative and sperm nuclei showed very weak or nulle signal (Fig. 2B-B'', C-C''). No immunofluorescence was observed in any other subcellular compartments.

After microspore reprogramming, the immunolocalization signal of acetylated histone H4 and H3 increased. Nuclei of proembryos and globular embryos displayed high fluorescent signals (Fig. 2D-D', 2E-E'). As embryogenesis progressed, immunofluorescence signal intensified in stages of heart-shaped (Fig. 3A-A', 3B-B'), and torpedo embryos (Fig. 3C-C'). Controls avoiding the first antibodies did not show significant labelling in any sample and developmental stage.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** The two developmental pathways of the microspore. Selected stages of *in vivo* pollen development (A-C) and stress-induced microspore embryogenesis (D-H) of *Brassica napus*. (A-G) Micrographs of semithin sections stained with toluidine blue and observed under bright field microscopy. (A) Vacuolated microspore, initial stage for both developmental pathways, pollen development and microspore embryogenesis. (B) Bicellular pollen. (C) Tricellular pollen. (D) Microspore-derived proembryo, still surrounded by exine. (E) Globular embryo. (F) Heart embryo. (G) Torpedo embryo. (H) Region of a Petri dish showing cotyledonary embryos, formed in culture after 30 days. Bars represent: (A-E): 10 µm, (F, G): 50 µm, (H): 1 mm.</figcaption></figure>
<figure><img src="image_5.png" /></figure>

(caption on next page)
**Fig. 2. Localization of acetylated histones during pollen development and early stages of microspore embryogenesis.** Immunolocalization of H3Ac and H4Ac during *in vivo* pollen development (A-A', B-B', C-C'') and stress induced microspore embryogenesis (D-D', E-E). Confocal microscopy images showing H3Ac or H4Ac immunofluorescence signal (green), DAPI staining of nuclei (blue) and Nomarsky's differential interference contrast (DIC). (A-A) H3Ac localization in vacuolated microspore. (B-B) H3Ac localization in tricellular pollen. (C, C) H4Ac localization in tricellular pollen. (D, D') H3Ac localization in globular embryo. (E, E) H4Ac localization in globular embryo. Bars represent: (A-A', B-B', C-C'') 10 µm, (D-D', E-E): 100 µm.

## 3.2. Changes in global H4 acetylation levels and HAT activity during microspore embryogenesis and pollen development

The immunofluorescence results and the intensity of labelling were similar for acetylated H3 and H4 in all the developmental stages analyzed, suggesting that the temporal profile of global levels of the two epigenetic marks would be analogous. Therefore, we selected the acetylated histone H4 for further quantification of its global level using ELISA assays. The quantification of acetylated histone H4 levels was performed during both microspore developmental programs. For the assays during *in vivo* pollen development, we selected the stages of vacuolated microspores and tricellular pollen, isolated directly from the anthers of floral buds. Additionally, for analysis during microspore embryogenesis, the stages of isolated vacuolated microspores, pro-embryos, and mature cotyledonary embryos were selected. The results revealed opposite patterns in the global levels of acetylated histone H4 along both developmental pathways.

During pollen development, a significant decrease in acetylated histone H4 levels was observed after differentiation and mature pollen grain formation (0.013 ± 0.002 OD) compared to the vacuolated microspore stage (0.043 ± 0.000 OD) (Fig. 4A). Conversely, following microspore reprogramming, the pattern of acetylated histone H4 exhibited an opposite pattern. An increase in histone H4 acetylation levels was observed in proembryos (0.051 ± 0.000 OD) compared to vacuolated microspores, with the highest histone H4 acetylation level reached at the cotyledonary embryo stage (0.129 ± 0.019 OD) (Fig. 4B).

Given that the global level of histone acetylation, both for histone H3 and histone H4, is determined by the activity of Histone Acetyl-transferase enzymes (HATs), a colorimetric enzymatic immunoassay was conducted based on the recognition of an acetylated substrate, subsequent to the transfer of the acetyl group from acetyl-CoA molecule and detected by a high-affinity antibody. The HAT enzymatic activity was assessed and compared between the two microspore developmental programs, analysing the aforementioned stages.

Results obtained from colorimetric detection, directly proportional to HAT enzymatic activity, revealed a distinct pattern in both developmental pathways (Fig. 4C, D). During *in vivo* pollen development, vacuolated microspores exhibited high levels of HAT activity (348.42 ± 105.07 ng acetyl CoA h⁻¹ mg protein⁻¹), whereas in the tricellular pollen stage HAT activity was similar than the negative control (Fig. 4C).

In microspore embryogenesis, following microspore reprogramming induced by stress, a significant increase in HAT enzymatic activity levels was detected in proembryos (875.53 ± 30.82 ng acetyl CoA h⁻¹ mg protein⁻¹) compared to the vacuolated microspore stage. The highest observed values of HAT enzymatic activity were in the differentiated cotyledonary embryos (1576.69 ± 16.93 ng acetyl CoA h⁻¹ mg protein⁻¹) (Fig. 4D).

## 3.3. Gene expression patterns of histone acetyltransferase *BnHAC5-like* during pollen development and microspore embryogenesis

Additionally, expression analysis of *BnHAC5-like*, a gene encoding a Histone Acetyltransferase (HAT), was performed using RT-qPCR. Histone acetylation is typically correlated with increased gene activity. To analyze the gene expression profile of a histone acetylation-modifying enzyme, we selected the *BnHAC5-like* gene. This selection was based on spatio-temporal gene expression data available in the Arabidopsis eFP browser (https://bar.utoronto.ca/gaia/search?query=hac5), which reported expression of this gene during both male gametophytic

development and zygotic embryo development. *BnHAC5-like* encodes a histone acetyltransferase capable of adding an acetyl group to both histone H3 and histone H4 lysines. It was analyzed during both microspore developmental pathways across the same aforementioned stages. Results obtained through qPCR showed a significant decrease in mRNA levels of the *BnHAC5-like* gene after pollen grain differentiation, in tricellular pollen (0.76 ± 0.05 a.u.) compared to vacuolated microspores, which served as the expression normalization reference (Fig. 5A).

In contrast, gene expression results during microspore embryogenesis development exhibited an opposite pattern. After vacuolated microspores reprogramming, the proembryo stage showed a significant increase (4.66 ± 0.95 a.u.) compared to vacuolated microspores. The highest gene expression levels of *BnHAC5-like* were observed at the cotyledonary embryo stage (12.75 ± 2.86 a.u.) (Fig. 5B).

## 3.4. Effects of the histone deacetylase inhibitor SAHA on microspore embryogenesis initiation

In order to analyse the role of histone acetylation in microspore reprogramming towards embryogenesis, treatments with SAHA, a specific inhibitor of classes I and II histone deacetylases (HDAC) (Drummond et al., 2005), were assayed on microspore cultures. Different concentrations of SAHA (1, 2.5 and 5 µM) were applied at the beginning of the culture and their effects on microspore induction were assessed by analysing the percentage of proembryos formed after 4 days.

Treatment with SAHA significantly increased proembryo production at 2.5 µM compared to control cultures (Fig. 6A). However, cultures treated with 1 and 5 µM SAHA showed a similar percentage of proembryos to that of untreated cultures. Considering the lack of effect of the drug at 1 and 5 µM concentration and the pronounced effect observed on microspore induction in the 2.5 µM SAHA-treated cultures, proembryos from the latter were selected for further analysis.

To verify the inhibitory effect of SAHA treatment on HDAC enzymes, changes in global histone H4 acetylation levels were analyzed on untreated and 2.5 µM SAHA-treated proembryos. The findings indicated that, in comparison with control proembryos (0051 ± 0000 O.D.), SAHA-treated proembryos exhibited nearly a six-fold increase in H4 acetylation levels (0229 ± 0034 O.D.) (Fig. 6B), indicating that the SAHA compound increased histone acetylation levels, probably by reducing HDAC activity.

## 3.5. Expression patterns of somatic embryogenesis-marker genes during microspore reprogramming

As embryogenesis induction is potentially controlled by epigenetic marks regulating gene expression (Horstman et al., 2017), and SAHA treatment induces increased embryogenesis initiation, we investigated whether histone hyperacetylation induced by SAHA regulates somatic embryogenesis-related genes during microspore reprogramming to embryogenesis. Among the numerous genes associated with embryogenesis, *LEC2*, *FUS3*, and *AGL15* are key regulators of plant cell totipotency, known for their roles in embryo identity and somatic embryogenesis induction (Méndez-Hernández et al., 2019; Ibañez et al. 2020).

We analyzed the expression levels of these transcription factor genes in vacuolated microspores before embryogenesis induction, and in proembryos. Results showed an important increase of the three genes in proembryos produced under control conditions (Fig. 7A, C, E). Then, we compared the expression in proembryos, in both untreated cultures and
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Localization of acetylated histones during advanced stages of microspore embryogenesis. Immunolocalization of H3Ac and H4Ac, confocal microscopy images showing H3Ac or H4Ac immunofluorescence signal (green) and Nomarsky's differential interference contrast (DIC). (A-A) H3Ac localization in globular to heart torpedo embryo. (B-B) H4Ac localization in heart-shaped embryo. (C, C) H4Ac localization in torpedo embryo. Bars represent 100 µm.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>B

**Global H4 Acetylation**</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C

**HAT Activity**

HAT activity (ng acetyl Co-A h-1 mg-1 protein)</figcaption></figure>

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

**HAT Activity**

HAT activity (ng acetyl Co-A h-1 mg-1 protein)</figcaption></figure>

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

**Fig. 4.** Patterns of histone H4 acetylation levels and histone acetyl transferase activity during pollen development and microspore embryogenesis. (A, B) Quantification of global H4 acetylation during *in vivo* pollen development (A) and stress-induced microspore embryogenesis (B). The bar charts show the changes in the amount of global H4 acetylation as revealed by ELISA-like test, indicated as optical density (OD) values at 450 nm. (C, D) Histone acetyl transferase (HAT) enzymatic activity during *in vivo* pollen development (C) and stress-induced microspore embryogenesis (D). The bar charts show the quantity of acetyl groups incorporated from the acetyl-CoA molecule. Bars on the columns represent the standard error of the mean (SEM), and different letters indicate significant differences among stages according to ANOVA and Tukey's test at *p* ≤ 0.05.

<figure><img src="image_11.png" /><figcaption>B
**BnHAC5-like**

Normalized expression (a.u.)

Vac. Microspore
a

Proembryo
b

Cotyledonary embryo
b</figcaption></figure>

<figure><img src="image_12.png" /></figure>

**Fig. 5.** Expression patterns of *BnHAC5-like* during pollen development and microspore embryogenesis. (A) Expression during pollen development. (B) Expression during microspore embryogenesis. mRNA levels are determined by qPCR and normalized to the vacuolated microspore stage. Bars on the columns represent the standard error of the mean (SEM), and different letters indicate significant differences among stages according to ANOVA and Tukey's test at *p* ≤ 0.05.

cultures treated with 2.5 µM SAHA. Under SAHA treatment, transcript levels of all genes exhibited increased expression compared to untreated proembryos (Fig. 7B, D, F). These results suggested that the inhibition of histone deacetylation by SAHA leads to the up-regulation of specific embryogenic genes, consequently triggering higher rates of microspore reprogramming.

4. DISCUSSION

Several epigenetic studies have demonstrated the importance of histone acetylation in different processes of plant development, such as seed germination, hypocotyl cell elongation, chlorophyll biosynthesis, meristem development and flowering, among others (Kumar et al., 2021; Ning et al., 2019; Zhao et al., 2019). Nevertheless, to date, there is a limited number of studies exploring the dynamics of histone
<figure><img src="image_13.png" /><figcaption>**Fig. 6.** Effects of SAHA treatment on microspore embryogenesis induction and histone H4 acetylation levels. (A) Embryogenesis induction efficiency, as indicated by the percentage of proembryos formed, in control and SAHA-treated cultures at various concentrations. Images show representatives areas of untreated and 2,5 µM SAHA-treated cultures; arrows point to proembryos. Scale bars in the images represent 50 µm. (B) Quantification of global H4 acetylation at the stage of proembryo in control and 2,5 µM SAHA-treated cultures, as revealed by ELISA-like test, indicated as optical density (OD) values at 450 nm. Bars on the columns represent the standard error of the mean (SEM), different letters indicate significant differences among stages according to ANOVA and Tukey's test at *p* ≤ 0.05.</figcaption></figure>

modifications associated with the change of the microspore fate from the in vivo normal developmental pathway towards an embryogenic route.

The aim of this research was to conduct a comparative analysis of the acetylation dynamics in histone proteins during the two developmental programs of the microspore: the *in vivo* pollen grain development and the *in vitro* stress-induced microspore embryogenesis. The purpose is to assess the potential role of histone acetylation throughout both developmental processes, evaluating its relevance at microspore embryogenesis induction in *Brassica napus*, a plant model system for analyzing induced cellular reprogramming. The vacuolated microspore stage, common in both developmental pathways and the starting point for embryogenesis induction, was selected as a reference stage in this comparative study, enabling a clear comparison of alterations in the dynamics of this epigenetic mark when diverting the microspore from its normal *in vivo* developmental pathway to an *in vitro* induced microspore embryogenesis after the application of an inductive stress.

### 4.1. Histone acetylation levels decrease during *in vivo* pollen development

In the present study, the localization of acetylated histone H3 and H4 was carried out using immunofluorescence techniques, along with the quantification of the global levels of acetylated histone H4 using an ELISA-like assay. The results from both experimental approaches were consistent and revealed a decrease in acetylation levels of histones as the pollen grain matures.

During the gametophytic development of *L. longiflorum*, a reduction in the acetylation levels of H4K5 and H4K8 has been demonstrated as the pollen grain maturation progresses (Janousek et al., 2000). The outcome of the global balance of histone epigenetic modifications is determined by the action of their corresponding modifying enzymes, which have been proposed as regulators of development and cellular reprogramming (Fehér, 2015). Among these, histone acetyltransferases (HATs) have been described as modulators of transcriptional activity in eukaryotic cells through the modification of the N-terminal end of residues, primarily lysines of histones H3 and H4, by adding an acetyl group from the coenzyme A molecule (Hu et al., 2019). It is established
A

BnFUS3

<figure><img src="image_14.png" /></figure>

B

BnFUS3

<figure><img src="image_15.png" /></figure>

C

BnAGL15

<figure><img src="image_16.png" /></figure>

D

BnAGL15

<figure><img src="image_17.png" /></figure>

E

BnLEC2

<figure><img src="image_18.png" /></figure>

F

BnLEC2

<figure><img src="image_19.png" /></figure>

Fig. 7. Effect of SAHA on expression patterns of the somatic embryogenesis transcription factors *BnFUS3*, *BnAGL15* and *BnLEC2*. (A, C, E) Gene expression in control cultures before (vacuolated microspore) and after embryogenesis induction (proembryo stage). (B, D, F) Gene expression in control and SAHA-treated cultures at the stage of proembryo formation, after embryogenesis induction. mRNA levels are determined by qPCR and normalized to the vacuolated microspore stage (A, C, E) and to the untreated (control) proembryos (B, D, F), respectively. Bars on the columns represent the standard error of the mean (SEM), and different letters indicate significant differences among stages according to ANOVA and Tukey's test at *p* ≤ 0.05.
that the relationship and balance between HAT and HDAC enzymes contribute to the control of numerous biological processes including embryo development, seed germination, and flowering, as well as the regulation of transcription under stress conditions (Boycheva et al., 2014; Cai et al., 2024). Furthermore, previous studies have described the dynamics of histone deacetylation, mainly of histones H3 and H4, during cell division in tobacco cell cultures (Li et al., 2005).

Given that they are the primary responsible for the global level of histone acetylation in chromatin fiber, in this study we evaluated the HAT enzymatic activity during pollen grain development. The results obtained correlated with the decreasing profile of global levels of histone H4 acetylation, as well as with the fluorescence intensity patterns obtained through the immunolocalization of acetylated histone H3 and H4.

Additionally, the gene expression of *BnHAC5-like* was analyzed. This gene encodes a histone acetyltransferase from the CBP family, responsible for adding an acetyl group to lysines K9 and K14 of histones H3 and H4, respectively. We selected this gene because data available in the Arabidopsis eFP browser (https://bar.utoronto.ca/gaia/search?query=hac5) about *BnHAC5-like* demonstrate that it is expressed during both male gametophytic development and zygotic embryo development. The results of this experiment revealed that *BnHAC5-like* decreased its expression during pollen grain differentiation, a pattern that correlates with the other dates we obtained during the gametophytic development of *Brassica napus*.

These results indicate that H3 and H4 histone acetylation accompanied early stages of pollen development in *B. napus*. However, as pollen maturation progresses, there is a negative regulation of the *BnHAC5-like* gene as well as a decrease in HAT activity and in global histone acetylation levels.

## 4.2. Stress-induced microspore reprogramming towards an embryogenic pathway involves an increase in histone acetylation levels

It is documented in the literature that histone acetylation in mammals is essential for proper embryogenic development, associated with the accessibility of protein complexes during DNA replication (Lagger et al., 2002; Vogelauer et al., 2000). In the field of plant biology, numerous cellular and molecular changes associated with the switch in developmental program, including the induction of *in vitro* embryogenesis, have also been described. Among these, epigenetic regulation has been reported during microspore embryogenesis as a crucial regulatory mechanism involved in cell reprogramming in various species such as *B. napus*, *H. vulgare*, and *T. aestivum* (El-Tantawy et al., 2014; Rodríguez-Sanz H et al., 2014; Seifert et al., 2016; Solís et al., 2015). In the study by Seifert et al. (2016), transcriptome sequencing through RNA-seq of different stages of *T. aestivum* microspore embryogenesis revealed 5269 transcripts differentially expressed during early embryogenesis. Furthermore, Gene Ontology enrichment analyses indicate that transcripts associated with metabolic and biosynthetic processes are down-regulated, while those related to chromatin modifications are up-regulated following inductive stress. This suggests that epigenetic regulation and chromatin remodelling are fundamental for microspore reprogramming towards embryogenic development in *T. aestivum*. These data are consistent with findings in the present study, where a significant increase in H4 histone acetylation has been detected upon microspore reprogramming, suggesting that the change in the developmental program of the microspore involved the chromatin remodelling.

While the correlation of the results of the AcH3 and AcH4 immunofluorescence, the quantification of AcH4 level and the HAT activity highly support a decrease in H3 and H4 acetylation levels during gametophytic development, data obtained in embryogenic development of the microspore have revealed an opposite dynamic. We observed that *in vitro*, following the application of inductive stress, the microspore reprogramming, the totipotency acquisition, and the initiation of

embryogenesis involve an increase in the acetylation levels of histones. An increment in the global levels of H4 acetylation was observed, which correlated with high levels of HAT activity and higher expression of *BnHAC5-like* gene. This increase was evident from the earliest divisions, in the proembryo stage, and shows the highest levels with embryo maturation, in the cotyledonary embryo stage. Previous studies have demonstrated that the switch in the microspore developmental program involves epigenetic modifications. This has been evidenced by observed changes in DNA methylation (Solís et al., 2012) and histone H3K9 methylation (Berenguer et al., 2017). With the new findings obtained in the present study, we further extend the evidence to include the involvement of histone acetylation during microspore reprogramming towards an embryogenic pathway.

All these results indicate that the epigenetic control through the acetylation of H3 and H4 histones plays an important role in the reprogramming and cell proliferation during microspore embryogenesis initiation in *B. napus*. The increase in histone acetylation would promote cellular reprogramming, as elevated levels in acetylated histone residues would lead to a less condensed chromatin conformation, favouring the transcriptional activity of genes involved in the microspore reprogramming.

## 4.3. Development and differentiation of microspore-derived embryos require high levels of histone H4 acetylation

In *B. napus*, after microspore reprogramming, the formed proembryos undergo a developmental pathway similar to zygotic embryogenesis in dicotyledonous plants, progressing through globular, heart, torpedo, and cotyledonary embryo stages (Prem et al., 2012). In this study, we analyzed developing and cotyledonary embryos and the results shown an increment in acetylated histones with embryo development progression. This increase was confirmed through immunolocalization assays, as well as by quantifying global H4 acetylation.

Numerous studies demonstrate the importance of histone modifications during cellular differentiation in plants (Furuta et al., 2011; He et al., 2012). At the genetic level, an increase in the expression of numerous genes encoding epigenetic modulators has been described during embryogenesis progression and embryo differentiation. For example, genes encoding argonaute proteins (AGO4 (ARGONAUTE 4), AGO5, AGO6), DNA methyltransferases (DDM1 (DECREASED DNA METHYLATION 1), DRM2, MET1, CMT3), histone methyltransferases (SUVH1, SUVR4, SUVR5) and histone acetyltransferases (HAC5) show increased expression during embryogenesis while some genes encoding DNA methyltransferases (DRM1) or histone deacetylases (HDA6 and HD19) reduce their gene expression levels (Seifert et al., 2016; Solís et al., 2012).

Our findings revealed the dynamism of histone acetylation not only during the early stages of microspore embryogenesis in response to stress and reprogramming, but also in more advanced stages of somatic embryogenesis. The results obtained in this work demonstrated higher expression levels of HAC5, specifically the *BnHAC5-like* gene, during microspore embryo development, with peak expression observed in the cotyledonary embryo stage. Although the involvement of other HAT genes cannot be excluded, our findings suggest the contribution of the analyzed enzyme in regulating the levels of histone acetylation during the embryogenic program of the microspore.

## 4.4. The histone deacetylase inhibitor SAHA enhances induction of microspore embryogenesis while increases expression of key somatic embryogenesis transcription factors

The relevance of histone acetylation during embryogenic reprogramming and cell proliferation and differentiation processes has been studied in diverse *in vitro* plant culture systems (Lee et al., 2016; Li et al., 2014; Rodríguez-Sanz H et al., 2014; Uddenberg et al., 2011). In the
present study we conducted functional analyses on isolated microspore cultures, treating them with SAHA, a specific histone deacetylase inhibitor that has been scarcely tested in plant species.

Previous studies have demonstrated the potential of other histone deacetylase inhibitors in cellular reprogramming. HDAC modulators, such as the Trichostatin A (TSA), have been employed in combination with the use of mutants to analyze the relevance of histone acetylation/deacetylation during various biological processes (Tanaka et al., 2008; Zhang et al., 2013). In animals, the histone deacetylase inhibitor TSA enhances the reprogramming of somatic cells during embryo cloning (Beigh et al., 2017; Miyamoto et al., 2017). In plants, TSA has been demonstrated to promote embryogenesis induction in species such as *A. thaliana*, *Picea abies*, *Pinus sylvestris* and *Vitis vinifera* (Abrahamsson et al., 2017; Tanaka et al., 2008; Uddenberg et al., 2011; Martínez et al. 2021), as well as plant regeneration in wheat (Bie et al. 2020). It has been also observed that TSA promote cellular totipotency in *B. napus* microspore cultures and promote embryo and green seedlings regeneration in *T. aestivum* microspore cultures (Jiang et al., 2017; Li et al., 2014).

Our analyses revealed that treatment with SAHA at a concentration of 2.5 µM leads to an increased number of proembryos and a significant elevation in global histone acetylation levels in cultures. These findings demonstrate for the first time that the histone deacetylase inhibitor, SAHA, also produces an increase in the percentage of microspore embryogenesis induction of *B. napus*. These results have particular interest because, although Trichostatin A maintains a similar micromolar potency, SAHA would be of great interest since it is almost ten times more cost-effective and has a broader affinity spectrum targeting the active sites of multiple HDAC classes (Methot et al., 2008). This may have contributed to the fact that, although TSA is widely used in basic research, SAHA has become the first HDAC inhibitor to receive approval from the Food and Drug Administration (FDA) for clinical use and it is being implemented in the treatment of certain cancer types (Bieliauskas et al., 2007; Duvic et al., 2007). Other HDAC inhibitor, less used in plants and less costly than TSA, sodium butyrate, has been tested in some vitro embryogenesis systems, however, the reports showed heterogeneous results depending on the system and species. It enhanced the embryogenic response in gravine (Martínez et al. 2020) and *B. rapa* (Zhang et al. 2016) *in vitro* cultures, while it did not provide any improvement in embryogenic cultures of wheat (Wang et al. 2019). Other reports have described a positive effect of this inhibitor on wheat plant regeneration, being a genotype-dependent effect (Bie et al. 2020).

Our findings highlight that the maintenance of high levels of histone acetylation in the early stages of microspore embryogenesis, modulated by the SAHA compound, promotes embryogenic induction through enhanced cellular reprogramming and totipotency acquisition. However, additional research is required to elucidate the precise molecular mechanisms regulating the activity of this compound during the process.

In both animals and plants, an open chromatin structure has been suggested as a prerequisite for maintaining the cell's totipotent state (Bosković et al., 2014; Burton and Torres-Padilla, 2010; Gaspar-Maia et al., 2011; González-Muñoz et al., 2014; Lu and Zhang, 2015; Pillot et al., 2010; Solís et al., 2015). It is described that after microspore reprogramming, the proembryos of different plant species show a decondensed chromatin pattern, a feature identified as crucial for proliferative cells (Bárany et al., 2005; Seguí-Símarro et al., 2011; Testillano et al., 2005, 2000).

Based on this, chromatin decondensation induced by the action of SAHA as a histone deacetylase inhibitor, could modify the transcription of genes associated with embryogenesis. To further probe SAHA effect, we evaluated the expression of three differentially expressed embryogenic-marker genes: *BnFUS3*, *BnAGL15*, and *BnLEC2*. FUSCA 3 (FUS3), LEAFY COTYLEDON 2 (LEC2), and AGAMOUSLIKE 15 (AGL15) have been identified as key transcription factors up-regulated during somatic embryogenesis induction in many plant species, including *B. napus* (Gulzar et al., 2020; Méndez-Hernández et al., 2019).

The results of this assay indicated an increase in the expression levels of these three embryogenesis marker genes after microspore reprogramming towards the embryogenic pathway, specifically at the proembryo stage. Furthermore, the expression of these genes was analyzed in proembryo samples from untreated and treated cultures with the SAHA inhibitor. This analysis revealed significantly higher expression levels in the SAHA-treated proembryos compared to the controls.

The findings of this study suggested that the increase in histone acetylation and the consequent reduction in chromatin condensation, is necessary for the microspore reprogramming and the totipotency acquisition. We demonstrate the positive effect of the deacetylase inhibitor SAHA in promoting embryogenesis initiation, which present a significant advantage, being ten times more cost-effective than Trichostatin A, the most commonly used histone deacetylase inhibitor in research publications of this field. These results complement previous findings from our group, wherein reduced chromatin condensation mediated by small molecule inhibitors of other epigenetic marks such as DNA methylation (El-Tantawy et al., 2014, Solís et al. 2015, Carneros et al. 2024) or histone H3K9 methylation (Berenguer et al., 2017) also favored embryogenesis induction. It has been demonstrated that in *Brassica napus*, DNA methylation levels increase during pollen development and decrease upon microspore embryogenic induction (Solís et al., 2012). This pattern is opposite to the epigenetic mark of histone acetylation that we have analyzed in the present study, which diminishes with pollen maturation and increases with microspore reprogramming towards embryogenesis. The dynamics of these two epigenetic marks also correlate with data obtained during other developmental processes in plant species such as chestnut, azalea, and pine (Meijón et al., 2010; Santamaría et al., 2011; Valledor et al., 2010).

### 4.5. *Opposite histone acetylation dynamics regulate the two developmental pathways and fates of the microspore*

Taking all the results together, we can suggest that microspore reprogramming towards an embryogenic pathway involves global changes in the genome-wide post-translational histone modifications. Specifically, an increase in histone acetylation levels is necessary for embryogenic induction. Furthermore, we have observed that these changes occurring with microspore reprogramming are contrary to those during *in vivo* pollen development in *B. napus*, where pollen grain maturation is linked to a decrease in the acetylation levels of H3 and H4 histones.

The current study demonstrates that during stress-induced microspore embryogenesis, treatment with the SAHA inhibitor enhances the efficiency of cellular reprogramming and embryogenesis initiation, along with an increase in histone acetylation. These findings reveal new opportunities that involve the use of pharmacological strategies with inhibitor compounds targeting histone epigenetic regulators, to improve the embryogenesis induction efficiency in crop species.

## Competing interests statement

The authors have no competing interests to declare.

## Author contributions

EB performed most of the experimental work. YPP and EC contributed to immunofluorescence and gene expression assays, and data analyses. YPP prepared the first draft of the manuscript. PST conceived, designed, and supervised the work, and revised the manuscript. All authors read and approved the final manuscript.

## Funding

Supported by Projects PID2020-113018RB-I00 and PID2023-146824OB-I00 (funded by MCIN/AEI /10.13039/
501100011033), TED2021-129633B-100 and CPP2021-008750 (funded by MCIN/AEI /10.13039/501100011033 and NextGenerationEU/PRTR).

## CRediT authorship contribution statement

**Yolanda Pérez-Pérez:** Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. **Elena Carneros:** Validation, Methodology, Investigation, Formal analysis, Data curation. **Eduardo Berenguer:** Validation, Methodology, Investigation, Formal analysis, Data curation. **Pilar S. S. Testillano:** Writing – review & editing, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## 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.

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