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.

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.

A broad spectrum of host plant responses to the actions of the gall midge: case study of Robinia pseudoacacia L. and Obolodiplosis robiniae (Haldeman).

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.

Temperature Regulation of Primary and Secondary Seed Dormancy in Rosa canina L.: Findings from Proteomic Analysis.

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.

Mitochondria Are Important Determinants of the Aging of Seeds.

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.

DNA synthesis pattern, proteome, and ABA and GA signalling in developing seeds of Norway maple (Acer platanoides).

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.