Plant Science

The plant cell wall is a dynamic and complex extracellular matrix that not only provides structural integrity and determines cell shape but also mediates intercellular communication. Among its major components, pectins play essential roles in cell adhesion, wall porosity, hydration, and flexibility. Rhamnogalacturonan-I (RG-I), a structurally diverse pectic polysaccharide, remains one of the least understood components of the plant cell wall. Its backbone is substituted with arabinan, galactan, and arabinogalactan side chains that vary in length, branching, and composition across tissues, species, and developmental stages. In addition, RG-I can undergo modifications such as backbone acetylation, further contributing to its structural complexity and functional diversity. To advance understanding of RG-I, we present a detailed method for isolating RG-I from the model plant Arabidopsis thaliana. Leveraging Arabidopsis as a model system provides major advantages owing to its well-characterized genome and powerful molecular toolkit, enabling deeper investigation into the roles of RG-I in plant development and responses to environmental stress. Our method consists of two major steps: an initial chemical extraction using oxalate, followed by endo-polygalacturonase (EPG) digestion to fragment the pectic domains. An advantage of this approach is that it produces a dry material that can be stored at room temperature without special handling and does not introduce chemicals that may interfere with downstream analyses. The purified RG-I can be used for detailed compositional and structural analyses, as well as for functional studies of enzymes involved in pectin biosynthesis, modification, and degradation. Although this protocol was developed for isolating RG-I from Arabidopsis rosette leaves, it is also applicable to other Arabidopsis organs and other plant species.

Rhamnogalacturonan-II (RG-II) is one of the least studied domains of pectin, primarily due to its low abundance, the lack of reliable antibodies, and the complexity of its structure. The present study builds upon existing protocols and procedures used to analyse RG-II in tissues where it is more abundant, combining and adapting them for the isolation of RG-II from Arabidopsis seed mucilage—a structure previously thought to lack RG-II. By applying these adapted methods, we first confirmed the presence of RG-II in seed mucilage and subsequently succeeded in isolating it from a tissue where it is typically present in low abundance, thereby enabling future studies on this previously overlooked component.

Starch is a carbohydrate widely used in the plant kingdom as a fuel for different physiological processes. While different techniques are available for the quantification of starch stored in seeds and bark tissues, they have hardly been used to quantify starch content in developing flower buds, where starch has been reported to accumulate in different reproductive organs. Here, we detail a quantitative enzymatic method to measure starch concentration in developing flower primordia in sweet cherry (Prunus avium L.). First, starch is enzymatically hydrolyzed to D-glucose, which was then quantified by an enzyme-coupled assay involving hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) and spectrophotometric quantification of NADH absorbance at 340 nm. This method is a sensitive, rapid, and affordable protocol specifically optimized for tiny flower buds with low starch content. The technique is revealed to successfully determine starch content in non-freshly harvested samples—frozen and stored at -20 °C or stored in fixatives—allowing a temporal separation of sampling and quantification and making the protocol suitable for high-throughput experimental designs in different fields of plant research.

Carbohydrates serve crucial functions in most living cells, encompassing structural and metabolic roles. Within the realms of plant and algal biology, carbohydrate biosynthesis and partitioning play pivotal roles in growth, development, stress physiology, and various practical applications. These applications span diverse fields, including the food and feed industry, bioenergetics (biofuels), and environmental management. However, existing methods for carbohydrate determination tend to be costly and time-intensive. In response to that, we propose a novel approach to assess carbohydrate partitioning from small samples. This method leverages the differential solubility of various fractions, including soluble sugars, starch, and structural polymers (such as cellulose). After fractionation, a straightforward spectrophotometric analysis allows for the quantification of sugars.

Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography–mass spectrometry (GC–MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC–MS.

Key features

• Comparative analysis of monosaccharide content in Arabidopsis-derived pectin between different genotypes or different treatments.

• Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue.

• Allows quick analyses of neutral and acidic monosaccharides simultaneously.

Graphical overview

Xyloglucan is one of the main components of the primary cell wall in most species of plants. This protocol describes a method to analyze the composition of the enzyme-accessible and enzyme-inaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks unsubstituted glucose residues in the backbone. The identities and relative amounts of released xyloglucan fragments are then determined using MALDI-TOF mass spectrometry.

In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.

The plant cell wall is primarily composed of the polysaccharides cellulose, hemicellulose and pectin. The structural and compositional complexity of these components are important for determining cell wall function during plant growth. Moreover, cell wall structure defines a number of functional properties of plant-derived biomass, such as rheological properties of foods and feedstock suitability for the production of cellulosic biofuels. A typical characterization of cell wall chemistry in the molecular biology lab consists of a mild acid hydrolysis for the quantification of hemicellulose and pectin-derived monomers and a separate analysis of cellulose by the Updegraff method. We have adopted a streamlined ‘one-step two-step’ hydrolysis protocol that allows for the simultaneous determination of cellulose content, neutral sugars, and uronic acids by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of paired samples. In our work, this protocol has largely replaced Updegraff cellulose quantification and hydrolysis with 2 M TFA for the determination of matrix polysaccharide composition at the micro scale.

The Arabidopsis thaliana seed coat epidermis produces copious amounts of mucilage polysaccharides (Haughn and Western, 2012). Characterization of mucilage mutants has identified novel genes required for cell wall biosynthesis and modification (North et al., 2014). The biochemical analysis of seed mucilage is essential to evaluate how different mutations affect cell wall structure (Voiniciuc et al., 2015c). Here we describe a robust method to screen the monosaccharide composition of Arabidopsis seed mucilage using ion chromatography (IC). Mucilage from up to 48 samples can be extracted and prepared for IC analysis within 24 h (only 4 h hands-on). Furthermore, this protocol enables fast separation (31 min per sample), automatic detection and quantification of both neutral and acidic sugars.

The Arabidopsis thaliana seed coat produces large amounts of cell wall polysaccharides, which swell out of the epidermal cells upon hydration of the mature dry seeds. While most mucilage polymers immediately diffuse in the surrounding solution, the remaining fraction tightly adheres to the seed, forming a dense gel-like capsule (Macquet et al., 2007). Recent evidence suggests that the adherence of mucilage is mediated by complex interactions between several cell wall components (Griffiths et al., 2014; Voiniciuc et al., 2015a). Therefore, it is important to evaluate how different cell wall mutants impact this mucilage property. This protocol facilitates the analysis of monosaccharides in sequentially extracted mucilage fractions, and quantifies the detachment of each component from seeds.