High-throughput method for Oxygen Consumption Rate measurement (OCR) in plant mitochondria.

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.

Redox dynamics in seeds of Acer spp: unraveling adaptation strategies of different seed categories.

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-h1, 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.

The chances in the redox priming of nondormant recalcitrant seeds by spermidine.

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.

Assessment of ITS2 Region Relevance for Taxa Discrimination and Phylogenetic Inference among Pinaceae.

The internal transcribed spacer 2 (ITS2) is one of the best-known universal DNA barcode regions. This short nuclear region is commonly used not only to discriminate taxa, but also to reconstruct phylogenetic relationships. However, the efficiency of using ITS2 in these applications depends on many factors, including the family under study. Pinaceae represents the largest family of extant gymnosperms, with many species of great ecological, economic, and medical importance. Moreover, many members of this family are representatives of rare, protected, or endangered species. A simple method for the identification of Pinaceae species based on DNA is necessary for their effective protection, authentication of products containing Pinaceae representatives, or phylogenetic inference. In this study, for the first time, we conducted a comprehensive study summarizing the legitimacy of using the ITS2 region for these purposes. A total of 368 sequences representing 71 closely and distantly related taxa of the seven genera and three subfamilies of Pinaceae were characterized for genetic variability and divergence. Intra- and interspecies distances of ITS2 sequences as well as rates of sequence identification and taxa discrimination among Pinaceae at various taxonomic levels, i.e., the species complex, genus, subfamily, and family, were also determined. Our study provides a critical assessment of the suitability of the ITS2 nuclear DNA region for taxa discrimination among Pinaceae. The obtained results clearly show that its usefulness for this purpose is limited.

New Insight into Taxonomy of European Mountain Pines, Pinus mugo Complex, Based on Complete Chloroplast Genomes Sequencing.

The Pinus mugo complex is a large group of closely related mountain pines, which are an important component of the ecosystems of the most important mountain ranges, such as the Alps, Carpathians and Pyrenees. The phylogenetic relationships between taxa in this complex have been under discussion for many years. Despite the use of many different approaches, they still need to be clarified and supplemented with new data, especially those obtained with high-throughput methods. Therefore, in this study, the complete sequences of the chloroplast genomes of the three most recognized members of the Pinus mugo complex, i.e., Pinus mugo, Pinus rotundata and Pinus uncinata, were sequenced and analyzed to gain new insight into their phylogenetic relationships. Comparative analysis of their complete chloroplast genome sequences revealed several mutational hotspots potentially useful for the genetic identification of taxa from the Pinus mugo complex. Phylogenetic inference based on sixteen complete chloroplast genomes of different coniferous representatives showed that pines from the Pinus mugo complex form one distinct monophyletic group. The results obtained in this study provide new and valuable omics data for further research within the European mountain pine complex. They also indicate which regions may be useful in the search for diagnostic DNA markers for the members of Pinus mugo complex and set the baseline in the conservation of genetic resources of its endangered taxa.