Plant Science

Seeds ensure the growth of a new generation of plants and are thus central to maintaining plant populations and ecosystem processes. Nevertheless, much remains to be learned about seed biology and responses of germinated seedlings to environmental challenges. Experiments aiming to close these knowledge gaps critically depend on the availability of healthy, viable seeds. Here, we report a protocol for the collection of seeds from plants in the genus Populus. This genus comprises trees with a wide distribution in temperate forests and with economic relevance, used as scientific models for perennial plants. As seed characteristics can vary drastically between taxonomic groups, protocols need to be tailored carefully. Our protocol takes the delicate nature of Populus seeds into account. It uses P. deltoides as an example and provides a template to optimize bulk seed extraction for other Populus species and plants with similar seed characteristics. The protocol is designed to only use items available in most labs and households and that can be sterilized easily. The unique characteristics of this protocol allow for the fast and effective extraction of high-quality seeds. Here, we report on seed collection, extraction, cleaning, storage, and viability tests. Moreover, extracted seeds are well suited for tissue culture and experiments under sterile conditions. Seed material obtained with this protocol can be used to further our understanding of tree seed biology, seedling performance under climate change, or diversity of forest genetic resources.

Key features

• Populus species produce seeds that are small, delicate, non-dormant, with plenty of seed hair. Collection of seed material needs to be timed properly.

• Processing, seed extraction, seed cleaning, and storage using simple, sterilizable laboratory and household items only. Obtained seeds are pure, high quality, close to 100% viability.

• Seeds work well in tissue culture and in experiments under sterile conditions.

• Extractability, speed, and seed germination were studied and confirmed for Populus deltoides as an example.

• Can also serve as template for bulk seed collection from other Populus species and plant groups that produce delicate seeds (with no or little modifications).

Graphical overview

Understanding the influence of secondary metabolites from fungi on the mitochondria of the host plant during infection is of great importance for the knowledge of fungus–plant interactions in general; it could help generate resistant plants in the future and in the development of specifically acting plant protection products. For this purpose, it must first be possible to record the mitochondrial parameters in the host plant. As of the date of this protocol, no measurements of mitochondrial respiration parameters have been performed in wheat paleae. The protocol shown here describes the measurements using the XF24 analyzer, which measures the rate of oxygen consumption in the sample by changes in the fluorescence of solid-state fluorophores. This procedure covers the preparation of samples for the XF24 analyzer and the measurement of mitochondrial parameters by adding specific mitochondrial inhibitors. It also shows the necessary approach and steps to be followed to obtain reliable, reproducible results. This is a robust protocol that allows the analysis of mitochondrial respiration directly in the wheat paleae. It demonstrates an important add-on method to existing screenings and also offers the possibility to test the effects of early infection of plants by harmful fungi (e.g., Fusarium graminearum) on mitochondrial respiration parameters.

Key features

• This protocol offers the possibility of testing the effects of early infection of plants by pathogens on mitochondrial respiration parameters.

• This protocol requires a Seahorse XF24 Flux Analyzer with Islet Capture Microplates and the Seahorse Capture Screen Insert Tool.

Graphical overview

Lipids in biomembranes can control the structure and, therefore, the functionality of membrane-embedded protein complexes. Unraveling how the lipid composition determines the mode of operation of membrane proteins provides mechanistic insights into their functionality. We applied a proteoliposome technique for studying how proteins function in biomembranes. The incorporation of isolated membrane proteins in preformed liposomes made from a well-defined lipid composition (proteoliposomes) is a powerful tool for studying lipid-protein interactions. Over several decades, the proteoliposome technique was employed for many different membrane proteins. Recently, it was recognized that different lipid compositions control the light-harvesting functionality of the major photosynthetic light-harvesting complex II (LHCII) isolated from plant thylakoid membranes in vitro. This technique allows systematic examination of the role of so-called non-bilayer lipids on light-harvesting characteristics of LHCII. This protocol describes the isolation of LHCII from leaves and details a four-step procedure to incorporate the detergent-solubilized membrane protein in large unilamellar vesicles (LUV). The protocol was optimized to ensure a very high lipid/protein ratio, designed to specifically examine lipid-protein interactions by minimizing LHCII aggregation. The procedure provides structurally and functionally highly intact LHCII in a detergent-free lipid bilayer with a defined composition.

Dark respiration refers to experimental measures of leaf respiration in the absence of light, done to distinguish it from the photorespiration that occurs during photosynthesis. Dark aerobic respiration reactions occur solely in the mitochondria and convert glucose molecules from cytoplasmatic glycolysis and oxygen into carbon dioxide and water, with the generation of ATP molecules. Previous methods typically use oxygen sensors to measure oxygen depletion or complicated and expensive photosynthesis instruments to measure CO₂ accumulation. Here, we provide a detailed, step-by-step approach to measure dark respiration in plants by recording CO₂ fluxes of Arabidopsis shoot and root tissues. Briefly, plants are dark acclimated for 1 hour, leaves and roots are excised and placed separately in airtight chambers, and CO₂ accumulation is measured over time with standard infrared gas analyzers. The time-series data is processed with R scripts to produce dark respiration rates, which can be standardized by fresh or dry tissue mass. The current method requires inexpensive infrared gas analyzers, off-the-shelf parts for chambers, and publicly available data analysis scripts.