Plant Science

Various environmental stresses or artificial reagents can trigger unfolded protein accumulation in the endoplasmic reticulum (ER) due to the folding capacity of the ER being exceeded. This is termed ER stress, and triggers the unfolded protein response (UPR). Assays for activation of the UPR in plants include Tunicamycin (Tm)- or dithiothreitol (DTT)-mediated root growth inhibition, analysis of splicing of the UPR-responsive transcription factor bZIP60 (basic Leucine Zipper Domain 60), and upregulation of relevant UPR genes. We provide here a quick and robust method to detect UPR signaling in Arabidopsis thaliana protoplasts. This assay can also be applied to other plant species for which protoplasts can be isolated.

This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g., comparing different gene expression levels in guard cells versus whole leaf to determine if a gene of interest is predominantly expressed in guard cells. It can also be used to study the effect of treatments or different genetic backgrounds in the regulation of the guard cell expressed genes.

Zygotes of the Fucale species are a powerful model system to study cell polarization and asymmetrical cell division (Bisgrove and Kropf, 2008). The Fucale species of brown algae grow in the intertidal zone where they reproduce by releasing large female eggs and mobile sperm in the surrounding seawater. The gamete release can be induced from sexually mature fronds in the laboratory and thousands of synchronously developing zygotes are easily obtained. In contrast to other eukaryotic models, such as land plants (Brownlee and Berger, 1995), the embryo is free of maternal tissues and therefore readily amenable to pharmacological approaches. The zygotes are relatively large (up to 100 µm in diameter), facilitating manipulations and imaging studies. During the first hours of zygote development, the alignment of the axis to external cues such as light is labile and can be reversed by light gradients from different directions. A few hours before rhizoid emergence, the alignment of the axis and the polarity are fixed and the cells germinate accordingly. At this stage the zygotes are naturally attached to the substratum through the secretion of cell wall adhesive materials (Kropf et al., 1988; Hervé et al., 2016). The first cell division occurs about 24 h after fertilisation and the early embryo is composed of only two cell types that differ in size, shape and developmental fates (i.e., thallus cells and rhizoid cells) (Bouget et al., 1998). The embryo can be successfully cultivated in the laboratory for a few more days (4 weeks maximum) and has an invariant division pattern during the early stages, which allows cell lineages to be traced histologically.

Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare Lilium davidii or Oryza sativa plasma membrane with high purity.

Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al., 2009; Mustroph et al., 2009) and ribosome footprinting (Ingolia et al., 2009; Ingolia et al., 2013). With this protocol, we have been able to successfully isolate and analyze high-quality ribosomal footprints from different stages of in vitro grown Arabidopsis thaliana plants (dark-grown seedlings [Merchante et al., 2015] and 13-day-old plantlets in plates and plants grown in liquid culture [unpublished results]).

Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. This protocol, adapted from Zhang et al. (2011), describes a procedure for the isolation of rice protoplasts from green tissue and shows an efficient and rapid method for isolation of nuclei form these protoplasts which are commonly used in a variety of experimental procedures including the isolation of high-molecular-weight DNA (Watson and Thompson, 1986), in vitro DNA synthesis (Roman, 1980), isolation of labeled transcripts for differential screening of cDNA libraries (Somssich et al., 1989), preparation of nuclear extracts for in vitro transcription systems (Roberts and Okita, 1991), isolation of nuclear proteins (Harrison et al., 1992) and studies of protein targeting to the nucleus (Hicks and Raikhel, 1993).

Plant transformation and exogenous protein expression is essential for molecular biology and biotechnology. Current approaches of stable plant transformation might be problematic and very time-consuming. Because of this, transient expression in protoplasts has become valuable alternative, being less cost and time-effective at the same time. Excellent for eukaryotic proteins, representing a natural cell habitat, protoplast isolation is widely used in protein interaction visualization techniques, like BiFC (Bimolecular fluorescence complementation) and FRET (Förster resonance energy transfer). In this protocol we present a another use of Arabidopsis protoplast in protein degradation assay, proving its high versatility as a tool in proteomics.

The protoplasts assay constitutes a powerful tool that allows an easy uptake of active agents and a precise quantification of cell death induction in different populations. Our study showed that the basal level of cell death in our controls is low and stable throughout the length of our experiments (Danon et al., 2005; Pineau et al., 2013). In addition, the data obtained from the protoplast assay are applicable to intact seedlings, where it is possible to see differences in the intensity of necrotic lesions (Danon et al., 2006) even if those differences are not as easily and clearly quantifiable as with the protoplast assay.

A lot of transcriptional profiling in plant and animals has used RNAs samples from many different cell types. The laser-capture microdissection (LCM) can identify and harvest pure cellular populations directly from heterogenous tissues based on histological identification. The molecules or protein isolated from LCM-captured cells can be suitable for single cell type analysis by using chip expression profiling or sequencing.