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.

Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 (Wang et al., 2020). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro enzymatic assay of magnesium chelatase (Wang et al., 2020). Currently, the crystallization studies of the BCM1 are underway. This protocol could be adapted to purify BCM1-like transmembrane proteins from eukaryotic organisms for enzymatic and structural studies.

The ureides allantoin and allantoate are the main organic nitrogen compounds transported in several legumes, predominantly from N₂ fixation. Moreover, recent studies point out a remarkable role for allantoin during several stress responses of plants other than legumes. The goal of this protocol is to determine ureides concentration in different plant tissues. Ureides are extracted from plant material by boiling it in phosphate buffer. The allantoin and allantoate present in the supernatants are subjected to alkaline-acidic hydrolysis to glyoxylate. The glyoxylate is converted into glycoxylic acid phenylhydrazone, that is then oxidized to red-colored 1,5-diphenylformazan. The absorbance of supernatants is measured using a spectrophotometer at 520 nm. Ureides concentration can be inferred by using a glyoxylate calibration curve. Ureide quantification of different tissues of Arabidopsis thaliana and soybean plants were carried out following this protocol.

Nicotinamide adenine dinucleotide phosphate (NADP) synthesis requires nicotinamide adenine dinucleotide (NAD) kinase activity, substrate NAD and ATP. The NAD kinase responds to various environmental stimuli and its activity is regulated via various regulatory pathways, such as Ca²⁺-dependent and redox-dependent signals. Conventional in vitro NAD kinase assay has been useful to evaluate enzyme activity; however, recent reports revealed a dynamics of NADP pool (the sum of NADP⁺ and NADPH) under fluctuating light condition, indicating that the rate of NADP synthesis is not always determined by NAD kinase activity. Here, we developed a novel method for the estimation of chloroplastic NAD kinase activity by quantifying the changes in the NADP amounts in response to illumination. As our approach does not involve protein extraction, it saves time (compared to the in vitro assay), thereby allowing for a sequence of assays, and provides several clues in the investigation of regulatory mechanisms behind NADP synthesis under various environmental conditions.

Nitric oxide (NO), is a redox-active, endogenous signalling molecule involved in the regulation of numerous processes. It plays a crucial role in adaptation and tolerance to various abiotic and biotic stresses. In higher plants, NO is produced either by enzymatic or non-enzymatic reduction of nitrite and an oxidative pathway requiring a putative nitric oxide synthase (NOS)-like enzyme. There are several methods to measure NO production: mass spectrometry, tissue localization by DAF-FM dye. Electron paramagnetic resonance (EPR) also known as electron spin resonance (ESR) and spectrophotometric assays. The activity of NOS can be measured by L-citrulline based assay and spectroscopic method (NADPH utilization method). A major route for the transfer of NO bioactivity is S-nitrosylation, the addition of a NO moiety to a protein cysteine thiol forming an S-nitrosothiol (SNO). This experimental method describes visualization of NO using DAF-FM dye by fluorescence microscopy (Zeiss AXIOSKOP 2). The whole procedure is simplified, so it is easy to perform but has a high sensitivity for NO detection. In addition, spectrophotometry based protocols for assay of NOS, Nitrate Reductase (NR) and the content of S-nitrosothiols are also described. These spectrophotometric protocols are easy to perform, less expensive and sufficiently sensitive assays which provide adequate information on NO based regulation of physiological processes depending on the treatments of interest.

The analysis of chemical diversity in non-sterile rhizosphere soil has been a pressing methodological challenge for years. Rhizosphere-enriched chemicals (i.e., rhizochemicals) include root exudation chemicals, (microbial) breakdown products thereof, and de novo produced metabolites by rhizosphere-inhabiting microbes, all of which can play an important role in plant-soil interactions. The power and resolution of analytical methods and statistical analysis pipelines allow for better acquisition, separation and identification of rhizochemicals, thus providing unprecedented insight into the biochemistry underpinning plant-soil interactions. The current protocol describes a recently developed method to characterize rhizochemical profiles from plants, including crops, and is modular and customizable, allowing for application across a range of different plant-soil combinations. The protocol provides in-depth details about the experimental system for sample collection, data acquisition by liquid chromatography coupled to mass spectrometry, and analytical pipeline, which statistically selects for rhizochemicals by statistical comparison between metabolite profiles from plant-containing soil and plant-free soil. Moreover, the optional addition of chemical standards permits a semi-targeted approach, which improves the annotation of chemical signatures and identification of single rhizochemicals.

In plants, macroautophagy, here referred as autophagy, is a degradation pathway during which the double-membrane structure named autophagosome engulfs the cargo and then fuses with vacuole for material recycling.

To investigate the process of autophagy, transmission electron microscopy (TEM) was used to monitor the ultrastructure of autophagic structures and identify the cargo during this process due to its high resolution. Compared to other autophagy examination methods including biochemical assays and confocal microscopy, TEM is the only method that indicates the morphology of autophagic structures in nanoscale, which is considered to be one of the best ways to illustrate the morphology of autophagic intermediates and the substrate of autophagy. Here, we describe the autophagy examination assay using TEM in Nicotiana benthamiana leaf cells.