2.1 Plant material collection

European beech seeds, which are categorized as intermediate storage seeds, were collected in 2020 from individual trees growing in Kórnik Arboretum (western Poland, 52°14′35″N, 17°05′33″E). These seeds were then dehydrated to 10%. Each lot was packed into a plastic container and stored at 4 °C. Fully developed embryos of mature seeds were isolated from intact seeds and imbibed in water overnight prior to TRX h1 target protein identification. For the determination of thiol (SH) content, embryonic axes were obtained from two seed lots harvested from the same tree in Kórnik Arboretum: a freshly harvested lot and a lot stored for 15 years at −10 °C in closed plastic boxes at 8–9% water content. The Kórnik Arboretum is owned and managed by the Institute of Dendrology, Polish Academy of Sciences. As the plant material was collected on the premises of the host institution, no specific permits were required to access the field site or to collect the seed material used in this study.

2.2 Mutagenesis and heterologous production of His-tagged TRX h1

Protein expression and purification were conducted in accordance with established protocols described in [21]. Briefly, the coding sequence of TRX h1 (from Pisum sativum) was subjected to PCR mutagenesis via specific primers that facilitated the replacement of a cysteine residue with serine at the active site (see Table 1 for primer details). Then, the amplicon was cloned and inserted into the expression vector pETM-11 in which a His-tag was added to the N-terminal end of the protein. The Escherichia coli BL21 strain was then transformed with the resulting expression plasmid. The transformed bacteria were then cultivated at 37 °C until an O.D. 600 nm of 0.4 was obtained, at which point protein expression was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hours. The recombinant protein His-CXXS was purified by Co²⁺ affinity chromatography in accordance with the manufacturer's instructions (HiTrap Talon, Cytiva). P. sativum was used as a bait species because, at the time of the study, the European beech genome was in an early draft version, and PCR primers designed based on its sequences were unreliable, limiting its use for protein interaction analyses. This approach allowed us to exploit a well-characterized TRX h1 sequence from a related plant species to identify potential targets in European beech seeds.

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Table 1. Oligonucleotides used for directed mutagenesis and heterologous expression of TRX h1 in Escherichia coli. Restriction sites are underlined (PeahN, NotI; PeahC, BamHI).

https://doi.org/10.1371/journal.pone.0359622.t001

2.3 Protein study with LC-MS/MS analysis

2.4 Western blot

To assess the redox state of the TRX h1-target interactions, the His-CXXS-bound protein samples from the embryonic-axis extracts were resolved under non-reducing and reducing conditions. For the non-reducing condition, samples were prepared in SDS-PAGE loading buffer without a reducing agent, leaving disulfide-linked TRX h1-target complexes intact; for the reducing condition, 50 mM DTT was added to cleave the mixed-disulfide bonds and release free TRX h1. Protein samples (20 µg) were separated on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (Immobilon-P; Merck Millipore, Burlington, MA, USA) at 350 mA for 1 h using a Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% non-fat milk and incubated with a mouse anti-TRX h1 antibody at a 1:5000 dilution, followed by an anti-mouse IgG secondary antibody conjugated with horseradish peroxidase at a 1:5000 dilution. Bands were visualized by enhanced chemiluminescence Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) and imaged on a G:BOX Chemi XRQ system (Syngene).

2.5 Determination of total thiol content

Total thiol (SH) groups were quantified in the embryonic axes of freshly harvested seeds and of seeds stored for 15 years to determine whether long-term storage was associated with changes in the thiol redox status of European beech seed axes. Samples were extracted in 0.02 M EDTA. SH groups were assayed with Ellman's reagent (DTNB). 0.001 M DTNB in methanol was added to 20 µL of sample in a final volume of 200 µL and absorbance was read at 412 nm (Infinite 200 PRO, Tecan). Under these conditions the reaction mixture contained approximately 79% (v/v) methanol, in which proteins are largely precipitated; the assay therefore reports the pool of thiols accessible to DTNB rather than the complete protein thiol pool, and the values are given as total thiols without subtraction of an acid-soluble fraction. For each sample, a paired blank without DTNB (DTNB replaced by an equal volume of buffer) was run to correct for the intrinsic absorbance of the extract; the DTNB-free value was subtracted from the corresponding reading. Thiol concentrations were calculated from the slope of a reduced glutathione (GSH) standard curve (0–200 µM, prepared with DTNB; R² = 0.988), taking the reagent blank of the sample plate as the zero point. Total thiol content is expressed per embryonic axis as GSH equivalents, obtained from the in-well concentration by correcting for the ten-fold dilution of the extract in the assay, the extraction volume and the number of axes per replicate. Three biological replicates per lot, each comprising ten embryonic axes, were analyzed; means were compared by Welch's t-test.

2.6 Cross-species comparison of TRX h1 targets

The European beech TRX h1 targets were compared with those previously identified in the embryonic axes of Norway maple (Acer platanoides L.) and sycamore maple (Acer pseudoplatanus L.) using the same His-CXXS trapping approach [21]. Because the three species do not share a common annotated genome, proteins were matched across species at the level of protein annotation, after normalization of names (lowercasing and removal of redundant whitespace and trailing punctuation). Of the 161 beech targets, 158 carried unique annotations and were used for the comparison; the remaining three corresponded to additional accessions sharing an annotation already present in the list. Shared and species-specific targets were determined and visualized as a Venn diagram in R 4.5.3 [24] using the ggVennDiagram package, version 1.5.7[25].

3.1 Genetic information processing

We identified 66 proteins involved in genetic information processing with a particular focus on proteins that are potential targets of TRX h1. The dominant proteins were chaperones and heat shock proteins (HSPs) as well as proteins involved in protein synthesis, folding, transport, and degradation. Additionally, we identified proteins related to RNA metabolism and the cell cycle (S1 Table). These findings highlight the importance of TRX-mediated redox regulation in maintaining protein homeostasis and ensuring proper cellular functions in the embryonic axes of beech seeds.

3.2 Heat shock proteins and chaperones as thioredoxin targets

In our study, the beech seed axes targeted by TRX h1 included HSPs from the HSP70 and HSP90 families. These molecular chaperones are critical for ensuring proper protein folding, assembly, translocation, and degradation, particularly under stress conditions. Chaperones were also identified as potential targets for TRXs in studies on spinach and poplar [27] and in Escherichia coli [28]. The identification of these HSPs as thioredoxin targets suggests that redox regulation may play a crucial role in modulating their activity during both stress responses and normal cellular processes [29]. Initially recognized in response to heat shock [30], HSPs are now known to be upregulated by various environmental and physiological stresses such as cold, UV light, mechanical injury, tissue remodeling, and pathogen invasion. TRX-mediated redox modifications can modulate the function of these HSPs and increase their ability to manage cellular stress and maintain homeostasis [29]. The regulation of HSP90 by TRX has already been extensively described in humans and animals [e.g., [31–33]]. However, in plants, many questions remain regarding the importance of HSP90 regulation by TRX. This topic offers a broad scope for further research.

3.3 DnaJ homolog and ribosome-associated complex

Another significant TRX h1 target we identified is the DnaJ homolog subfamily C member 2-like protein. This protein acts both as a cytosolic chaperone and a nuclear chromatin regulator. As part of the ribosome-associated complex (RAC), it assists in the folding of nascent polypeptides, whereas in the nucleus, it regulates gene expression by facilitating the activation of polycomb-repressed genes. The identification of this protein as a TRX h1 target suggests that redox regulation may influence its dual roles in protein folding and gene regulation, particularly through TRX-mediated modifications that impact its interactions with other proteins involved in these processes. Another DnaJ protein from the C subfamily, member 9, was also identified as a target protein for TRXs in studies on human TRXs [34].

Similarly, NudC proteins and J-domain proteins (JDPs), which have also been identified as potential TRX h1 targets, are critical for protein quality control. The modulation of JDPs by TRX could impact their interactions with HSP70s and affect the broader protein-folding machinery within the cell [35].

3.4 Nascent-Polypeptide-Associated Complex (NAC)

The NAC, another potential TRX h1 target, is a heterodimeric complex associated with ribosomes. NAC is believed to protect nascent polypeptide chains as they exit the ribosome. TRX-mediated redox regulation of NAC may affect its role in protein translocation either by inhibiting or facilitating the transport of newly synthesized proteins to specific cellular compartments [36]. These findings suggest that TRX regulation of NAC could be crucial for maintaining protein homeostasis, particularly during stressful conditions that require rapid adjustment of protein synthesis and transport.

3.5 Protein Disulfide Isomerases (PDIs) and their redox regulation

In plants, protein disulfide isomerases (PDIs) are key enzymes that assist in the folding of nascent and misfolded proteins by catalyzing disulfide bond formation. Our identification of PDIs as TRX targets could be particularly important during heat stress and other conditions that induce the unfolded protein response (UPR); PDIs, including members of the PDI-M family in Arabidopsis, are upregulated in response to such stress [37]. The fact that PDIs have been found among TRX h1 targets raises the question of a putative redox regulation of these proteins in plants.

3.6 Ribosomal proteins, elongation factors, and initiation factors as thioredoxin targets

Among the identified potential TRX h1 target proteins were several ribosomal proteins, elongation factors, and initiation factors, which are key components of the translation process. These include 60S ribosomal L2, L12, P0, P1, P3-like proteins, elongation factor 1-beta, and eukaryotic translation initiation factors (eIF3D and eIF4C).

The identification of numerous translation-related proteins, chaperones, and stress-response proteins targeted by TRX h1 highlights the central role of redox regulation in controlling key cellular processes in beech embryonic axes. TRX h1-mediated modifications may allow the protein synthesis machinery of the embryo to be fine-tuned during critical stages of development and ensure that protein homeostasis is maintained even under fluctuating environmental conditions. Further research into the specific redox modifications of these proteins will be essential for understanding the full scope of the regulatory role of TRX h1 during seed development, storage and germination.

The methyl CpG-binding protein (MBD) binds to methylated DNA via its MBDs, which is a conserved sequence motif of 70 amino acids. To date, 13 putative MBD-containing genes (AtMBD1–13) have been identified in the model plant Arabidopsis thaliana [38]. The identification of the MBD protein, which is involved in chromatin remodeling and DNA methylation, suggests that TRX h1 may influence epigenetic regulation during seed maturation and storage. Epigenetic changes, including DNA methylation, have been shown to play a role in seed dormancy and stress responses and further emphasizes the importance of TRX h1 in modulating gene expression in response to environmental cues.

3.7 Cellular and environmental process signaling

In this category, a total of 15 proteins were identified that are primarily associated with various cellular processes. The main categories included proteins involved in the following: calcium signaling and transport, such as calreticulin (CRT) and calmodulin, metabolic and enzymatic regulation, cytoskeleton organization and cell movement, and proteins related to stress response and defense mechanisms. Additionally, some of the identified proteins play roles in embryonic development and protein synthesis regulation (S1 Table). Calcium serves as a key secondary messenger in cell signal transduction pathways in both plants and animals. Furthermore, it regulates various cellular functions by binding to specific calcium-binding proteins. One such protein that we identified in our study is calmodulin, which plays a crucial role in regulating plant growth and development and in resistance mechanisms to various biotic and abiotic stresses [39]. Calmodulins regulate the expression of HSP genes under stress conditions [40,41]. The identification of a calmodulin family protein is important because we have identified numerous HSPs, as described above.

Calreticulin (CRT) is found in the endoplasmic reticulum of both plant and animal cells where it functions as a ubiquitous molecular chaperone. The antioxidant role of plant calreticulin in alleviating oxidative stress and enhancing stress tolerance has been demonstrated in transgenic plants. The overexpression of calreticulin from Triticum aestivum has led to increased drought resistance in tobacco plants cultivated under limited water conditions [42,43]. Research has shown that the C-domain of TaCRT1 is crucial for its ability to tolerate various stresses in plants [42]. The findings from several antioxidant assays indicate that, in contrast with wild-type plants, transgenic plants with CRT overexpression have significantly elevated levels of antioxidant enzymes including superoxide dismutase (SOD) [42,43]. Interestingly, in the context of our results, we observed the presence of Cu/Zn SOD in the proteome of the target proteins of TRX h1. Studies indicate that in animal cells, the upregulation of CRT is correlated with an increase in the levels of TRXs and their related antioxidant enzymes at the protein and mRNA levels [43–45]. In plants, this relationship between CRTs and TRXs has not been well described, although CRTs have previously been identified as target proteins for TRXs in Medicago truncatula seeds [46].

Translationally Controlled Tumor Proteins (TCTPs) in plants play diverse roles in stress tolerance [47], act as chaperone-like proteins in thermotolerance [48], aid in calcium binding [49], and contribute to oxidative stress defense [50]. Their ability to interact with proteins and protect against denaturation, as well as their involvement in calcium-mediated signaling pathways, highlights their importance in responding to abiotic stresses such as drought and heat [51]. TCTPs also show potential in biotic stress responses including an improved tolerance to pests and viruses [52]. Nevertheless, the role of TRX h1 in regulating TCTP activity in plants remains unknown.

3.8 Energy metabolism

In our study, we identified 47 TRX target proteins related to metabolism in European beech seed axes, which play essential roles in energy metabolism, particularly through the regulation of carbohydrate, amino acid, and lipid metabolic pathways. TRXs modulate the activity of key enzymes through redox-dependent changes and influence pathways critical for energy production, growth, and stress responses [27,53,54]. The identified target proteins highlight the extensive influence of TRX across various metabolic processes. In carbohydrate metabolism, several TRX-regulated enzymes, such as fructose-bisphosphate aldolase (FBA), malate dehydrogenase (MDH), and phosphoglycerate kinase (PGK), are essential for glycolysis and the TCA cycle, which are vital for energy production [16,55]. For example, FBA has been identified as a redox-sensitive protein in sycamore seeds, where oxidative modifications can influence its glycolytic activity, especially under dehydration stress. In sycamore seeds, the lack of “metabolic shutdown” due to oxidative stress could lead to continuous metabolic activity even under low-water conditions, which would increase the risk of energy depletion and reduce seed viability [55]. In contrast, Norway maple seeds exhibit tighter metabolic control, which likely contributes to their desiccation tolerance and capacity for long-term storage. During dormancy in Norway maple seeds, abscisic acid (ABA) downregulates FBA and potentially limits energy availability and delays germination [56]. MDH, another TRX h1 target, catalyzes the oxidation of malate in the TCA cycle and serves as a critical component in cellular respiration. Several proteomic studies suggest that mitochondrial MDH is a TRX target in plants [27,53,54]. In addition, recent studies have identified MDH as a TRX h1 target in the seed axes of sycamore (Acer pseudoplatanus), which further supports the role of TRXs in regulating energy metabolism during seed development and storage [21]. In sycamore seeds, MDH levels are modulated by endogenous gibberellins and ABA during dormancy release, which suggests a complex regulatory interplay between redox status and hormone signaling [57]. Similar findings occurred in Coffea arabica and Coffea eugenioides, where MDH expression decreases in desiccation-tolerant seeds and this supports our results on MDH regulation in beech seeds [58]. In amino acid metabolism, TRX regulates enzymes such as 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase, glycine cleavage system H protein, and adenosylhomocysteinase, which are involved in amino acid biosynthesis and degradation. This regulation ensures a balanced supply of amino acids, which are essential for protein synthesis, cellular growth, and the response to stress. Amino acid availability is crucial during seed development and germination, where metabolic flexibility can support resilience under fluctuating environmental conditions [59]. In lipid metabolism, TRX influences enzymes, such as acyl carrier proteins and AAA superfamily proteins, both of which are involved in lipid biosynthesis and membrane integrity. Through these enzymes, TRX helps maintain lipid homeostasis, which is essential for both energy storage and structural stability, particularly as seeds undergo dehydration and later rehydration during germination. Proper lipid regulation is crucial to ensure that seed membranes remain functional and allows seeds to recover from the desiccation required for long-term storage [60].

3.9 Redox regulation and oxidative stress

Our study revealed that key proteins, such as glutathione peroxidase (GPX) and glutaredoxin (GRX), interact with TRX h1 and contribute to redox homeostasis in European beech seed axes. These proteins play a central role in mitigating oxidative stress, which is a critical factor in seed aging and viability loss during storage [61]. GPX reduces harmful peroxides, whereas GRX maintains a protein thiol balance that protects cellular proteins from oxidative damage, which can lead to protein aggregation and functional loss [62]. The interactions of TRX h1 with these proteins suggest that enhancing the redox balance could improve seed longevity [21].

SODs, particularly those from the Cu/Zn-SOD family, serve as a first line of defense against ROS by converting superoxide radicals into hydrogen peroxide [63]. In beech seeds, SOD activity has been linked to ROS detoxification during storage with increased enzyme levels noted under stress conditions. However, excessive ROS levels can overwhelm these defenses and lead to a loss of viability, particularly under prolonged or extreme storage conditions [61].

The results of a study by [64] further emphasize the importance of superoxide metabolism during germination and growth and suggests that SOD activity and subsequent hydrogen peroxide production are essential for early cell wall modification and the initiation of seed germination.

The approximately 2.2-fold decrease in total thiol content per axis indicates that the axes shift towards a more oxidized state during storage. This change reflects oxidation of reactive cysteine residues, which may arise from reversible disulfide formation, S-glutathionylation and irreversible higher oxidation, potentially including cysteine residues within some of the redox-sensitive candidate TRX h1 targets identified here (e.g., GAPDH, FBA, MDH, GPX, GRX, Cu/Zn-SOD). The reversibly oxidized fraction is the natural substrate of the TRX h1 system, whereas irreversibly oxidized thiols lie beyond its reach. This is consistent with the recognized role of the protein thiol redox state as a marker of storage and desiccation tolerance in seeds [62] and points to a possible contribution of TRX h1-dependent redox maintenance to beech seed longevity. However, these measurements reflect the overall oxidative/reductive environment of the embryonic axes during storage and do not by themselves demonstrate a specific role for a particular thioredoxin; further functional studies are required to confirm the involvement of TRX h1 in maintaining seed viability.

This interpretation is further supported by our previous study on European beech seeds, in which protein-bound thiols were shown to be highly dynamic during development and storage. In that work, non-protein thiols remained comparatively stable during storage, whereas protein thiols declined, indicating that the protein thiol pool represents a particularly storage-sensitive component of the redox system in beech seeds [16]. The present results extend this earlier observation by linking storage-related changes in the total reduced thiol pool with the identification of candidate TRX h1-interacting proteins involved in metabolism, antioxidant defense and protein homeostasis.

In our previous studies, we identified TRX h1 target proteins in the embryonic axes of Norway maple and sycamore maple [21]. Therefore, we compared our new findings from European beech with TRX h1 targets across seeds with different desiccation tolerance strategies as follows: Norway maple (orthodox), sycamore maple (recalcitrant), and European beech (intermediate). Notably, the following six TRX h1 target proteins were conserved across all three species: phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate-dependent), Cu/Zn SOD, nucleoside diphosphate kinase, GPX, 60S acidic ribosomal protein P0, and a nonspecific lipid-transfer protein. Additionally, European beech shares 32 TRX h1 targets with only sycamore maple and three with only Norway maple (Fig 4), which reflects potential differences in protein redox regulation linked to desiccation tolerance strategies. The greater number of shared targets with the recalcitrant sycamore maple may suggest that intermediate seeds, such as European beech, retain redox mechanisms that are more aligned with desiccation-sensitive species. This finding provides valuable insights into the evolution of redox regulation across different seed categories and their potential adaptation to varying environmental conditions.

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Fig 4. Venn diagram illustrating the number of shared and unique proteins identified as TRX h1 targets in the embryonic axes of seeds from three tree species: European beech, Norway maple, and sycamore maple.

The diagram presents a synthesis of data from this study combined with previously published findings by Fuchs et al. [21].

https://doi.org/10.1371/journal.pone.0359622.g004