Acetylene Reduction Assay: A Measure of Nitrogenase Activity in Plants and Bacteria.

Nitrogen is one of the most abundant elements in the biosphere, but its gaseous form is not biologically available to many organisms, including plants and animals. Diazotrophic microorganisms can convert atmospheric nitrogen into ammonia, a form that can be absorbed by plants in a process called biological nitrogen fixation (BNF). BNF is catalyzed by the enzyme nitrogenase, which not only reduces N2 to NH3 , but also reduces other substrates such as acetylene. The acetylene reduction assay (ARA) can be used to measure nitrogenase activity in diazotrophic organisms, either in symbiotic associations or in their free-living state. The technique uses gas chromatography to measure the reduction of acetylene to ethylene by nitrogenase in a simple, quick, and inexpensive manner. Here, we demonstrate how to: prepare nodulated soybean plants and culture free-living Azospirillum brasilense for the ARA, use the gas chromatograph to detect the ethylene formed, and calculate the nitrogenase activity based on the peaks generated by the chromatograph. The methods shown here using example organisms can be easily adapted to other nodulating plants and diazotrophic bacteria. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Acetylene reduction assay in root nodules Basic Protocol 2: Acetylene reduction assay using diazotrophic bacteria Basic Protocol 3: Calculation of nitrogenase activity Support Protocol 1: Production of acetylene from calcium carbide Support Protocol 2: Calibration of the gas chromatograph Support Protocol 3: Total protein quantification.

High-Resolution Translatome Analysis Reveals Cortical Cell Programs During Early Soybean Nodulation.

Nodule organogenesis in legumes is regulated temporally and spatially through gene networks. Genome-wide transcriptome, proteomic, and metabolomic analyses have been used previously to define the functional role of various plant genes in the nodulation process. However, while significant progress has been made, most of these studies have suffered from tissue dilution since only a few cells/root regions respond to rhizobial infection, with much of the root non-responsive. To partially overcome this issue, we adopted translating ribosome affinity purification (TRAP) to specifically monitor the response of the root cortex to rhizobial inoculation using a cortex-specific promoter. While previous studies have largely focused on the plant response within the root epidermis (e.g., root hairs) or within developing nodules, much less is known about the early responses within the root cortex, such as in relation to the development of the nodule primordium or growth of the infection thread. We focused on identifying genes specifically regulated during early nodule organogenesis using roots inoculated with Bradyrhizobium japonicum. A number of novel nodulation gene candidates were discovered, as well as soybean orthologs of nodulation genes previously reported in other legumes. The differential cortex expression of several genes was confirmed using a promoter-GUS analysis, and RNAi was used to investigate gene function. Notably, a number of differentially regulated genes involved in phytohormone signaling, including auxin, cytokinin, and gibberellic acid (GA), were also discovered, providing deep insight into phytohormone signaling during early nodule development.

Soybean Hairy Root Transformation: A Rapid and Highly Efficient Method.

New genetic engineering techniques have advanced the field of plant molecular biology, and Agrobacterium-mediated transformation has enabled the discovery of numerous molecular and genetic functions. It has been widely used in many plants, including the economically important crop, soybean. Large-scale genetic analyses are needed to comprehend the molecular mechanisms that underlie the agronomic traits of soybean, and the generation of stable transgenic plants involves a lengthy and laborious process. Agrobacterium rhizogenes-mediated hairy root transformation is a quick and efficient method for investigations of root-specific processes and interactions. Generation of composite plants with transgenic roots and wild-type shoots allows for the study of the genetic mechanisms involved in root biology, such as the Bradyrhizobium-soybean interaction. Here, we provide an updated protocol for generating hairy soybean roots in as little as 18 days in a cost- and space-effective manner and demonstrate possible uses of composite plants with soybean nodulation assays and gene expression analysis as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Soybean hairy root transformation Basic Protocol 2: Soybean nodulation assay Alternate Protocol: Soybean nodulation assay in germination pouches Support Protocol: Bradyrhizobium japonicum culture preparation for inoculation Basic Protocol 3: Histochemical GUS staining for promoter analysis.