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Background: Seeds of woody plant species, such as those in the Acer genus like Norway maple ( Acer platanoides L.) and sycamore ( Acer pseudoplatanus L.), exhibit unique physiological traits and responses to environmental stress. Thioredoxins (Trxs) play a central role in the redox regulation of cells, interacting with other redox-active proteins such as peroxiredoxins (Prxs), and contributing to plant growth, development, and responses to biotic and abiotic stresses. However, there is limited understanding of potential variations in this system between seeds categorized as recalcitrant and orthodox, which could provide insights into adaptive strategies., Methods: Using proteomic analysis and DDA methods we investigated the Trx-h1 target proteins in seed axes. We complemented the results of the proteomic analysis with gene expression analysis of the Trx-h 1, 1- Cys-Prx , and TrxR NTRA genes in the embryonic axes of maturing, mature, and stored seeds from two Acer species., Results and Discussion: The expression of Trx-h1 and TrxR NTRA throughout seed maturation in both species was low. The expression of 1- Cys-Prx remained relatively stable throughout seed maturation. In stored seeds, the expression levels were minimal, with slightly higher levels in sycamore seeds, which may confirm that recalcitrant seeds remain metabolically active during storage. A library of 289 proteins interacting with Trx-h1 was constructed, comprising 68 from Norway maple and 221 from sycamore, with distinct profiles in each seed category. Recalcitrant seed axes displayed a wide array of metabolic, stress response, and signaling proteins, suggesting sustained metabolic activity during storage and the need to address oxidative stress. Conversely, the orthodox seed axes presented a protein profile, reflecting efficient metabolic shutdown, which contributes to their extended viability. The results of the study provide new insights into seed viability and storage longevity mechanisms. They enhance the understanding of seed biology and lay the foundation for further evolutionary research on seeds of different categories., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fuchs, Staszak, Vargas, Sahrawy, Serrato, Dyderski, Klupczyńska, Głodowicz, Rolle and Ratajczak.)

Global warming has adversely affected Picea abies (L.) H. Karst. forests in Europe, prompting the need for innovative forest-breeding strategies. Somatic embryogenesis (SE) offers promise but requires protocol refinement. Understanding the molecular mechanisms governing somatic embryo development is essential, as oxidative stress plays a crucial role in SE regulation. Ascorbic acid (ASA), is a vital antioxidant that can potentially control oxidative stress. In the present study, we normalized ASA concentrations in induction and proliferation media to enhance embryogenic tissue (ET) regeneration and proliferation capacity of mature explants. The media were supplemented with ASA at 0 mg l -1 , 25 mg l -1 , 50 mg l -1 , 100 mg l -1 , and 200 mg l -1 . The accumulation of hydrogen peroxide (H 2 O 2 ) and endogenous phytohormones, including auxins, cytokinins, brassinosteroids, abscisic acid, and gibberellin, was measured in non-embryonic calli and ET. Subsequently, their impact on ET induction and multiplication was analyzed. Our results demonstrate that application of ASA at concentrations of 25 mg l -1 and 200 mg l -1 led to increased H 2 O 2 levels, potentially inducing oxidative stress while simultaneously reducing the levels of all endohormone groups. Notably, the highest ET induction frequency (approximately 70%) was observed for ASA at 50 mg l -1 . These findings will enhance SE induction procedures, particularly in more resistant explants, underscoring the significance of ASA application to culture media., Competing Interests: The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hazubska-Przybył, Obarska, Konecka, Kijowska-Oberc, Wawrzyniak, Piotrowska-Niczyporuk, Staszak and Ratajczak.)

Background: Conventional methods to measure oxygen consumption, such as Clark-type electrodes, have limitations such as requiring a large amount of starting material. Moreover, commercially available kits for high-throughput methods are usually optimized for animal cells and mitochondria. Here, we present a novel method to measure the oxygen consumption rate using a high-throughput assay in isolated mitochondria of European beech seeds. To perform the measurements, we adapted the Agilent Seahorse XF Cell Mito Stress Test Kit protocol for measurements on plant mitochondria., Results: The optimized protocol for OCR measurement of mitochondria isolated from beech seeds allowed the observation of storage period-dependent gradual decreases in non-phosphorylating respiration, phosphorylating respiration and maximal FCCP-stimulated respiration. The longer the seeds were stored, the greater the impairment of respiratory function., Conclusions: Thanks to this method it is possible to minimize the amount of plant material and conduct research to obtain information on the respiratory condition and activity of plant mitochondria, including the efficiency of oxidative phosphorylation and the maximum oxidative capacity of the respiratory chain. We demonstrated that the improved protocol is suitable for study of plant material., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The problems posed by seed sensitivity to desiccation and aging have motivated the development of various techniques for mitigating their detrimental effects. The redox priming of seeds in antioxidant solution to improve their postharvest performance is one of the approaches. Spermidine (Spd) was tested as an invigorating solution on nondormant recalcitrant (desiccation-sensitive) seeds of the silver maple (Acer saccharinum L.). The treatment resulted in an 8-10% increase in germination capacity in seeds subjected to mild and severe desiccation, while in aged seeds stored for 6 months, no significant change was observed. The cellular redox milieu, genetic stability, mitochondrial structure and function were investigated to provide information about the cellular targets of Spd activity. Spermidine improved the antioxidative capacity, especially the activity of catalase, and cellular membrane stability, protected genome integrity from oxidative damage and increased the efficiency of mitochondria. However, it also elicited a hydrogen peroxide burst. Therefore, it seems that redox priming in nondormant seeds that are highly sensitive to desiccation, although it positively affected desiccated seed performance, may not be a simple solution to reinvigorate stored seeds with a low-efficiency antioxidant system., (© The Author(s) 2023. Published by Oxford University Press.)

This study aims to provide insights into plant-insect interaction during the formation and development of open gall structure on the leaves of Robinia pseudoacacia during gall formation by Obolodiplosis robiniae. This was the first time such far-reaching studies were performed at a biochemical and anatomical level. The gall wall is created from a few thick cells covered with epidermis. This parenchymatous nutritive tissue is rich in starch. Sclerenchyma only occurs around the vascular bundles as a result of the lignification of the parenchyma of the bundle sheaths. The level of reactive oxygen species (ROS) in the new structure was reduced and catalase activity was inhibited, which suggests another pathway of ROS decomposition - e.g. by ascorbate or glutathione peroxidase. The gall structure was combined with an increasing level of protein and non-protein thiols. Phenols seems to be a good protective factor; whose level was lower in infected leaflets. Levels of MUFA (monosaturated fatty acids) and SFA (saturated fatty acids) rose, probably as source of food for insects. The amount of fatty acid is positively correlated with the plant response. We detected that non infected leaflets produced C6:0 (hexanoic acid) and C8:0 (octanoic acid) fatty acids connected with odor. Changes in gall color as they develop are connected with photosynthetic pigments degradation (mainly chlorophylls) where the pathway of astaxanthin transformation to fatty acid is considered to be the most important process during gall maturation. Nutritive tissue is composed mainly of octadecanoic acid (C18:0) - a main source of food for O. robiniae., (© 2022. The Author(s).)

Plant metal hyperaccumulators, to which Brassica juncea belongs, must have very efficient defence mechanisms that enable growth and development in an environment polluted with various heavy metals. B. juncea (Indiana mustard) v. Małopolska was exposed to the activity of trace elements such as cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) in combinations: CuPb, CuCd, CuZn, PbCd, PbZn, and ZnCd in a concentration of 25 μM each for 96 h during control cultivation. We observed a clear tendency for metal uptake and accumulation in above-ground parts which is characteristic of hyperaccumulators. The combinations of CuCd, CuZn, and PbCd inhibited the development of the seedlings the most. The used metal combinations increased the levels of reactive oxygen species (ROS) such as: hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 .- ) and oxidized proteins in B. juncea organs, generating oxidative stress conditions in the cells. We determined the level of transcription of the respective defence proteins of the detoxification and antioxidant systems. We have shown that in the first 24 h of stress condiction, activation of glutamylcysteine-γ synthetase (yECS) and glutathione reductase (GR1) enzymes related to the detoxification of heavy metals is important for B. juncea plants. In addition, the data provide important information on how plants respond to the presence of heavy metals in the first days of stress conditions., (© 2021. The Author(s).)

Exposure to lead (Pb) can have serious toxic effects on the physiological and biochemical processes of plants. The chemical form of the metal determines the degree of its toxicity. In our research, we examined the effect of lead in the form of lead nitrate [Pb(NO3)2] and lead chloride (PbCl2) in concentrations of 12.5 mM and 25 mM on pine (Pinus sylvestris) seed germination. Nitrogen salt causes more severe changes than chloride salt. Increasing levels of electrolyte leakage, malondialdehyde, and hydrogen peroxide were detected during germination processes. The high levels of ROS lead to redox changes in the cell. We observed a reduction in the level of the reduced form of glutathione (GSH), and at the same time observed increased levels of the oxidised form of glutathione (GSSG) depending on the concentration and also the time of exposure to lead compounds. At the beginning of germination processes, the effective non-enzymatic activity of the antioxidant cycle was dominant, and at the late stage the enzymatic activity was noticed in the presence of Pb compounds. CAT activity significantly increased after Pb compound exposition., Competing Interests: The authors have declared that no competing interests exist.

Temperature is a key environmental factor restricting seed germination. Rose ( Rosa canina L.) seeds are characterized by physical/physiological dormancy, which is broken during warm, followed by cold stratification. Exposing pretreated seeds to 20 °C resulted in the induction of secondary dormancy. The aim of this study was to identify and functionally characterize the proteins associated with dormancy control of rose seeds. Proteins from primary dormant, after warm and cold stratification (nondormant), and secondary dormant seeds were analyzed using 2-D electrophoresis. Proteins that varied in abundance were identified by mass spectrometry. Results showed that cold stratifications affected the variability of the highest number of spots, and there were more common spots with secondary dormancy than with warm stratification. The increase of mitochondrial proteins and actin during dormancy breaking suggests changes in cell functioning and seed preparation to germination. Secondary dormant seeds were characterized by low levels of legumin, metabolic enzymes, and actin, suggesting the consumption of storage materials, a decrease in metabolic activity, and cell elongation. Breaking the dormancy of rose seeds increased the abundance of cellular and metabolic proteins that promote germination. Induction of secondary dormancy caused a decrease in these proteins and germination arrest.

The Pinus mugo complex includes several dozen closely related European mountain pines. The discrimination of specific taxa within this complex is still extremely challenging, although numerous methodologies have been used to solve this problem, including morphological and anatomical analyses, cytological studies, allozyme variability, and DNA barcoding, etc. In this study, we used the seed total protein (STP) patterns to search for taxonomically interesting differences among three closely-related pine taxa from the Pinus mugo complex and five more distant species from the Pinaceae family. It was postulated that STP profiling can serve as the backup methodology for modern taxonomic research, in which more sophisticated analyses, i.e., based on the DNA barcoding approach, have been found to be useless. A quantitative analysis of the STP profiles revealed characteristic electrophoretic patterns for all the analyzed taxa from Pinaceae. STP profiling enabled the discrimination of closely-related pine taxa, even of those previously indistinguishable by chloroplast DNA barcodes. The results obtained in this study indicate that STP profiling can be very useful for solving complex taxonomic puzzles.

Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Brassica species are known to display hyperaccumulation capability. Brassica juncea (Indiana mustard) v. Malopolska plants were exposed to trace elements, i.e., cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn), at a concentration of 50 μM and were then harvested after 96 h for analysis. We observed a high index of tolerance (IT), higher than 90%, for all B. juncea plants treated with the four metals, and we showed that Cd, Cu, Pb, and Zn accumulation was higher in the above-ground parts than in the roots. We estimated the metal effects on the generation of reactive oxygen species (ROS) and the levels of protein oxidation, as well as on the activity and gene expression of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The obtained results indicate that organo-specific ROS generation was higher in plants exposed to essential metal elements (i.e., Cu and Zn), compared with non-essential ones (i.e., Cd and Pb), in conjunction with SOD, CAT, and APX activity and expression at the level of encoding mRNAs and existing proteins. In addition to the potential usefulness of B. juncea in the phytoremediation process, the data provide important information concerning plant response to the presence of trace metals.

Seeds enable plant survival in harsh environmental conditions, and via seeds, genetic information is transferred from parents to the new generation; this stage provides an opportunity for sessile plants to settle in new territories. However, seed viability decreases over long-term storage due to seed aging. For the effective conservation of gene resources, e.g., in gene banks, it is necessary to understand the causes of decreases in seed viability, not only where the aging process is initiated in seeds but also the sequence of events of this process. Mitochondria are the main source of reactive oxygen species (ROS) production, so they are more quickly and strongly exposed to oxidative damage than other organelles. The mitochondrial antioxidant system is also less active than the antioxidant systems of other organelles, thus such mitochondrial 'defects' can strongly affect various cell processes, including seed aging, which we discuss in this paper., Competing Interests: The authors declare that they have no conflict of interests.

Mature seeds of Norway maple exhibit desiccation tolerance and deep physiological dormancy. Flow cytometry, proteomics, and immunodetection have been combined to investigate seed development of this species. DNA content analysis revealed that cell cycle/endoreduplication activity differs between seed organs and developmental stages. In the embryo axis, the proportion of the nuclei with the highest DNA content (4C) increases at the beginning of maturation (17 weeks after flowering; WAF), and then is stable until the end of maturation, to increase again after drying. In cotyledons, during maturation endopolyploid nuclei (8C) occur and the intensity of endoreduplication increases up to 21 WAF, and then is stable until development is completed. In dry mature seeds, the proportion of 4C nuclei is high, and reaches 36% in the embryo axis and 52% in cotyledons. Proteomic studies revealed that energy and carbon metabolism, fatty acid biosynthesis, storage and antioxidant proteins are associated with seed development. Study of the ABI5 protein, a transcription factor involved in ABA signalling, and the RGL2 protein, a repressor of the GA signalling indicates that the highest accumulation of these proteins occurs in fully-matured and dried seeds. It is suggested that this increase in accumulation can be associated with completion of maturation, mainly with desiccation and dormancy acquisition.

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower ( Brassica oleracea var. botrytis ) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation., Competing Interests: The authors declare no conflicts of interest.

Acer pseudoplatanus seeds are characterized by a deep physiological embryo dormancy that requires a few weeks of cold stratification in order to promote germination. Understanding the function of proteins and their related metabolic pathways, in conjunction with the plant hormones implicated in the breaking of seed dormancy, would expand our knowledge pertaining to this process. In this study, a proteomic approach was used to analyze the changes occurring in seeds in response to cold stratification, which leads to dormancy release. In addition, the involvement of abscisic (ABA) and gibberellic acids (GA) was also examined. Fifty-three proteins showing significant changes were identified by mass spectrometry. An effect of ABA on protein variation was observed at the beginning of stratification, while the influence of GA on protein abundance was observed during the middle phase of stratification. The majority of proteins associated with dormancy breaking in the presence of only water, and also ABA or GA, were classified as being involved in metabolism and genetic information processing. For metabolic-related proteins, the effect of ABA on protein abundance was stimulatory for half of the proteins and inhibitory for half of the proteins. On the other hand, the effect on genetic information processing related proteins was stimulatory. GA was found to upregulate both metabolic-related and genetic information processing-related proteins. While seed dormancy breaking depends on proteins involved in a variety of processes, proteins associated with methionine metabolism (adenosine kinase, methionine synthase) and glycine-rich RNA binding proteins appear to be of particular importance., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The proteome of zygotic embryos of Acer platanoides L. was analyzed via high-resolution 2D-SDS-PAGE and MS/MS in order to: (1) identify significant physiological processes associated with embryo development; and (2) identify changes in the proteome of the embryo associated with the acquisition of seed dormancy. Seventeen spots were identified as associated with morphogenesis at 10 to 13 weeks after flowering (WAF). Thirty-three spots were associated with maturation of the embryo at 14 to 22 WAF. The greatest changes in protein abundance occurred at 22 WAF, when seeds become fully mature. Overall, the stage of morphogenesis was characterized by changes in the abundance of proteins (tubulins and actin) associated with the growth and development of the embryo. Enzymes related to energy supply were especially elevated, most likely due to the energy demand associated with rapid growth and cell division. The stage of maturation is crucial to the establishment of seed dormancy and is associated with a higher abundance of proteins involved in genetic information processing, energy and carbon metabolism and cellular and antioxidant processes. Results indicated that a glycine-rich RNA-binding protein and proteasome proteins may be directly involved in dormancy acquisition control, and future studies are warranted to verify this association.