Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems.

Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.

Bacterial Shoot Apical Meristem Inoculation Assay.

By virtue of their sessile nature, plants may not show the fight-and-flight response, but they are not devoid of protecting themselves from disease-causing agents, attack by herbivores, and damages that are caused by other environmental factors. Plants differentially protect their life-sustaining organs such as plant apexes from the attack by microbial pathogens. There are well-established methods to inoculate/infect various plant parts such as leaves, roots, and stems with various different pathogens. The plant shoot apical meristems (SAM) are a high-value plant target that provides niche to stem cell populations. These stem cells are instrumental in maintaining future plant progenies by giving birth to cells that culminate in flowers, leaves, and stems. There are hardly few protocols available that allow us to study immune dynamics of the plant stem cells as they are hindered by various layers of the SAM cell populations. Here, we describe a step-by-step method on how to inoculate the Arabidopsis SAM with model plant pathogen Pseudomonas syringae pv. tomato DC3000.

Molecular Modeling of the Interaction Between Stem Cell Peptide and Immune Receptor in Plants.

Molecular docking enables comprehensive exploration of interactions between chemical moieties and proteins. Modeling and docking approaches are useful to determine the three-dimensional (3D) structure of experimentally uncrystallized proteins and subsequently their interactions with various inhibitors and activators or peptides. Here, we describe a protocol for carrying out molecular modeling and docking of stem cell peptide CLV3p on plant innate immune receptor FLS2.

Mapping a Transcriptome-Guided Arabidopsis SAM Interactome.

The advent of multi-OMICS approaches has a significant impact on the investigation of biological processes occurring in plants. RNA-SEQ, cellular proteomics, and metabolomics have added a considerable ease in studying the dynamics of stem cell niches. New cell sorting approaches coupled with the labeling of stem cell population specific marker genes are highly instrumental in enriching distinct cellular populations for various types of analysis. One more promising field of OMICS is the mapping of cellular interactomes. The plant stem cells research is barely profited from this newly emerging field of OMICS. Generation of stem cell/niche-specific interactome is a time-consuming and labor-intensive task. Here, we describe a method on how to assemble a SAM-based interactome after using the available generic Arabidopsis interactomes. To define the context of SAM in a generic interactome, we used SAM cell population transcriptome datasets. Our step-by-step protocol can easily be adopted for other stem cell niches such as RAM and lateral meristems keeping in view the availability of transcriptome datasets for cellular populations of these niches.