Hydroponic culture systems are widely used in research due to their intrinsic properties, such as easily altering nutrient composition, applying chemical treatments like metal, salt and hormones in the growth media, and collecting the root sample. Here, we describe a relatively easy and economic hydroponic culture system of the model plant Arabidopsis thaliana. It is worthy to note that this simple system can be adjusted and is suitable for other plant organisms.

This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any endogenous full-length transcript from the target gene. CRISPR-Trap is applicable for any target gene with no or little coding sequence in its first exon. Several human cell lines and different genes have so far been edited successfully with CRISPR-Trap.

This is a protocol for quantitative determination of storage and total carbohydrates in algae and cyanobacteria. The protocol is simple, fast and sensitive and it requires only few standard chemicals. Great advantage of this protocol is that both storage and total saccharides can be determined in the cellular pellets that were already used for chlorophyll and carotenoids quantification. Since it is recommended to perform the pigments measurement in triplicates, each pigment analysis can generate samples for both total saccharide and glycogen/starch content quantification.

The protocol was applied for quantification of both storage and total carbohydrates in cyanobacteria Synechocystis sp. PCC 6803, Cyanothece sp. ATCC 51142 and Cyanobacterium sp. IPPAS B-1200. It was also applied for estimation of storage polysaccharides in Galdieria (IPPAS P-500, IPPAS P-507, IPPAS P-508, IPPAS P-513), Cyanidium caldarium IPPAS P-510, in green algae Chlorella sp. IPPAS C-1 and C-1210, Parachlorella kessleri IPPAS C-9, Nannochloris sp. C-1509, Coelastrella sp. IPPAS H-626, Haematococcus sp. IPPAS H-629 and H-239, and in Eustigmatos sp. IPPAS H-242 and IPPAS C-70.

Aphids constitute a large group of Hemipterans that use their slender stylets to tap into the sieve elements of plants from which they consume copious amounts of phloem sap, thus depriving the plant of photoassimilates. Some aphids also transmit viral diseases of plants. Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is a polyphagous insect with a host range that covers 50 plant families, is considered amongst the top 3 insect pest of plants. The interaction between Arabidopsis thaliana and the GPA is utilized as a model pathosystem to study plant-aphid interaction. Here we describe the protocol used in our laboratories for rearing the GPA, and no-choice and fecundity bioassays to study GPA performance on Arabidopsis. In addition, we describe the procedure for the electrical penetration graph (EPG) technique to monitor feeding behavior of the GPA on Arabidopsis.

This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments.


Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli

Nitrate reductase (NR) reduces the major plant nitrogen source, NO₃^-, into NO₂^-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.

DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore the original RRBS protocol is not suitable for plants. Accordingly, we developed an in silico pipeline to select specific enzymes to generate a region of interest (ROI)-enriched, e.g., promoter-enriched, reduced representation genome in plants (Hsu et al., 2017). By digesting the maize genome with MseI and selecting 40-300 bp segments, we sequenced about one-fourth of the maize genome while preserving 84.3% of the promoter information. The protocol has been successfully established in maize and can be broadly used in any genome. Our in silico pipeline is combined with the RRBS library preparation protocol, allowing for the computational analysis and experimental validation.

Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the tissue. Here we describe a protocol to utilize micro-computed tomography (micro-CT) scanning to visualize vascular networks in the maize stem. The protocol covers sample fixation and staining with contrasting reagents, data acquisition using micro-CT, reconstructing three-dimensional (3D) models of stem inner structures and extraction of vascular networks from the model. This protocol can be easily applied to various types of species and organs/tissues.

In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al., 2017).

To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant E-tagged proteins). Laboratory-synthesis of gold nanoparticles has been previously described (Yang et al., 2011). Desired E-tagged proteins can be cloned from the corresponding genes, and expressed and purified using standard laboratory procedures. We will use E-tagged green fluorescent protein (GFP) as a reference protein here. Users can simply insert an E-tag into their protein of interest, at either terminus. To achieve maximum delivery efficiency, we suggest users testing different length of E-tags. For example, we inserted E = 0 to 20 (E0 means no E-tag insertion, and E20 means 20 glutamic acids insertion in a row) to most of the proteins we tested, and screened for optimal E-tagged length for highest delivery efficiency. E10-tagged proteins gave us the highest delivery efficiency for most of the proteins (except for Cas9, where E20 tag showed highest delivery efficiency).

Once these materials are ready, it takes about ~10 min to make the E-tagged protein and nanoparticle nanoassemblies, which are immediately used for delivery. Complete delivery (~100% for GFP-E10) is achieved in less than 3 h.

The opening of stomata in plants in response to blue light is driven by the plasma membrane H⁺-ATPase in guard cells. To evaluate the activation of the H⁺-ATPase in vivo, we can use H⁺-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of guard cell protoplasts and measure H⁺-pumping in the protoplasts. It is also necessary to determine the protein amount of H⁺-ATPase. In this protocol, we describe the procedures required for these preparations and measurements.