Plant Science

The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol purple, treated with the peptide and photographed after 30 min. Herein the protocol is optimized for evaluation of exogenous treatment, described in detail and expected results are presented.

Plant plasma membrane H⁺-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H⁺ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic activity of the plasma membrane H⁺-ATPase from Arabidopsis thaliana leaves.

Calcium plays important roles in maintaining plant cellular structure and also acts as a key secondary messenger in intercellular signaling. Thirty years ago, methods of detecting calcium in sub-cellular level had been established (Stockwell and Hanchey, 1982; Borgers et al., 1982) and reviewed extensively (Wick and Heplerm, 1982). We had used the method of testing calcium localization in salt tolerance improved transgenic alfalfa plant (Zhang and Wang, 2015). Here, we describe the protocol of testing calcium deposition by staining with potassium pyroantimonate (PPA) in detail, which was adapted from former reports (Stockwell and Hanchey, 1982; Borgers et al., 1982). The principle of this protocol is that the Ca²⁺ can react with antimonite and from black granules, which can be observed under a transmission electron microscope. The protocol includes common micromanipulation techniques of plant tissue, observation with a transmission electron microscope and photography.

Rice plants release proton (H⁺) from root cells into rhizosphere area leading to the acidification of the rhizosphere and increased solubility of ferric iron complexes on the cell membrane, which is important for iron uptakes. Here, we present a detailed protocol to measure H⁺ flux in root hairs of transgenic rice seedlings and transgenic rice protoplasts by the Non-invasive Micro-test Technique (NMT). The NMT system is based on a non-invasive microelectrode technology that is automatically controlled by a computer, to achieve a three-dimensional, real-time, dynamic characterization of the concentration, velocity, and direction of a variety of molecules or ions. Because there is no need to directly contact the measured cells that could cause cell damage, we are able to obtain accurate and real time information on ion concentration. This is the first protocol that describes the non-invasive micro measurement technique of both root hairs and protoplasts in rice. In NMT, voltage differences are measured at two excursion points that are manipulated using a computer. Voltage differences can be converted into H⁺ fluxes using the ASET 2.0 (The imFlux^® software) and JCal v3.2.1 Software. Analysis of the H⁺ fluxes provides a simultaneous measure of the crossing of a localized region of the root surface in response to stress, which provides real-time in-situ detection of net ion transport across membranes. This method will promote use of NMT in plant biology.

Heterologous expression of genes in budding yeast Saccharomyces cerevisiae (S. cerevisiae) is especially suitable to functionally study the corresponding encoded protein at the cellular level (Bonneaud et al., 1991). This is mainly because many strains defective in specific activities are available and could be complemented by homologous genes existing across the eukaryotic kingdom (http://www.yeastgenome.org/). However, the protocol we describe here is not a complementation but a “gain-of-function” assay. It is based on a drop-test assay that we have set up to assess the cellular zinc tolerance conferred by the expression of heterologous genes in the wild-type S. cerevisiae. Different dilutions of a yeast culture expressing the heterologous gene of interest are grown on a range of zinc-enriched plates, and are then compared to the control yeast expressing the empty vector. Working with different concentrations of both yeast and zinc are essential to succeed in describing zinc tolerance phenotype upon yeast transformation (Mirouze et al., 2006). This test has also proven to be valuable to differentiate among related members of gene families as exemplified for Arabidopsis Plant Defensin type1 (Shahzad et al., 2013).

Silicon (Si) is a biologically important element for plants in the order Poales (Yamamoto et al., 2011; Kido et al., 2015). In rice, Si is mainly deposited in the motor cells and the cell walls of the leaf epidermis. However, the molecular basis of this overall process has not been elucidated. Thus, we propose a protocol for the histochemical staining of the silica body based on specific hydrogen bonding between silanol group and the carboxylate group of crystal violet lactone (Ichimura et al., 2008), as described by Isa et al. (2010), but with minor modifications. This modified protocol can be used for observing Si accumulation during rice development.

This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction):

NH₄⁺ + hypochlorite + OH^- + phenol → indophenol

The oxidation of indophenol caused by phenol oxidation is a blue dye that is quantified at 635 nm in a spectrophotometer. Per ammonium molecule one molecule of indophenol is formed. The protocol described here is for Arabidopsis thaliana (A. thaliana) leaves and roots, although it is also valid for other plants species.

Accumulation of metals in plant tissues, and occasionally, different cells of the same tissue, may be highly non-uniform (Seregin and Kozhevnikova, 2008). Easy-to-use histochemical methods may greatly help to investigate the distribution and accumulation of metals within and among plant tissues, and also provide information on their subcellular localization (Seregin and Kozhevnikova, 2011). The histochemical techniques of zinc (Zn) visualization are based on the formation of the blue-colored complex of Zn with the metallochrome indicator Zincon (C₂₀H₁₅N₄NaO₆S), or the green-fluorescent complex with Zinpyr-1 (C₄₆H₃₆Cl₂N₆O₅) (Seregin et al., 2011; Seregin and Kozhevnikova, 2011). A method for histochemical Zn detection in plant tissues using Zinpyr-1 was first proposed by Sinclair et al. (2007), and later modified by Seregin et al. (2011), and Seregin and Kozhevnikova (2011). Histochemical data supplement the results of quantitative analysis, thus allowing a detailed study of the distribution, accumulation, and translocation pathways of Zn within the plant, which are important topics in modern plant physiology. These histochemical techniques have been successfully applied in different plant species, for example Zea mays (Seregin et al., 2011), Noccaea caerulescens and Thlaspi arvense (Kozhevnikova et al., 2014a), Capsella bursa-pastoris and Lepidium ruderale (Kozhevnikova et al., 2014b), in which Zn was detected in different root and shoot tissues. Here, we present the full staining protocols for these methods, developed or modified in our lab (Seregin and Kozhevnikova, 2011; Kozhevnikova et al., 2014a; Kozhevnikova et al., 2014b).

Potassium is known as a rate-limiting factor for crop yield and plays an important role in plants response under abiotic stresses. Recently, cytosolic K⁺ retention ability in leaf mesophyll has emerged as an important component of plant salt tolerance mechanism (Wu et al., 2013; Wu et al., 2014; Wu et al., 2015). In this protocol, the procedure for screening leaf mesophyll for K⁺ retention by the MIFE (microelectrode ion flux estimation) technique is described in detail using wheat as an example. By measuring NaCl-induced K⁺ efflux in leaf mesophyll, a large number of plant accessions can be screened and categorised according to their salinity stress tolerance. The method provides a rapid and reliable tool that targets the activity of specific membrane transporters directly contributing to salinity tolerance trait and, because of this, has a competitive advantage over traditional whole-plant phenotyping. While the focus of this protocol is on wheat, the suggested method may be adopted for screening K⁺ retention in leaf mesophyll in any other crop species.

Cytoplasmic calcium ([Ca²⁺]_cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca²⁺]_cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca²⁺]_cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca²⁺]_cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.