Plant Science

Recordings of electric potential changes on plant surfaces have been utilized to identify the components and mechanisms involved in the formation and transmission of systemic signals elicited by stimuli such as herbivory, wounding, or burning. The recorded responses, commonly referred to as slow wave or variation potentials, exhibit striking variability in their waveform. The extent to which this variability is due to differences in experimental procedures or plant biological variability remains unclear. Here, we provide a detailed and robust protocol refined from years of experience in conducting leaf surface potential recordings of Arabidopsis thaliana in response to mechanical wounding. This protocol serves as a comprehensive tutorial covering plant growth, procedures for reproducible mechanical wounding, critical aspects of electrophysiological recordings, and statistical analysis of surface potential recordings. It particularly emphasizes the construction and maintenance of electrodes, placement of the reference or ground electrode, mechanisms for wounding, and data analysis. This protocol aims to promote and facilitate the adoption, standardization, and interoperability of plant surface potential recordings among research groups, thereby increasing the reproducibility and comparability of data within the field.

MicroRNAs (miRNA) are small (21–24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves.

Graphical abstract

Plant nanobiotechnology is a flourishing field that uses nanomaterials to study and engineer plant function. Applications of nanotechnology in plants have great potential as tools for improving crop yield, tolerance to disease and environmental stress, agrochemical delivery of pesticides and fertilizers, and genetic modification and transformation of crop plants. Previous studies have used nanomaterials functionalized with chemicals, including biocompatible polymers with charged, neutral, or hydrophobic functional groups, to improve nanomaterial uptake and localization in plant cells. Recently, the use of biorecognition motifs such as peptides has been demonstrated to enable the targeted delivery of nanoparticles in plants (Santana et al., 2020). Herein, we describe a bio-protocol to target nanoparticles with chemical cargoes to chloroplasts in plant leaves and assess targeting efficiency using advanced analytical tools, including confocal microscopy and elemental analysis. We also describe the use of isothermal titration calorimetry to determine the affinity of nanomaterials for their chemical cargoes. Nanotechnology-based methods for targeted delivery guided by conserved plant molecular recognition mechanisms will provide more robust plant bioengineering tools across diverse plant species.

Graphic abstract:

Targeted delivery of nanomaterials with chemical cargoes to chloroplasts enabled by plant biorecognition