Protein SUMOylation is a key modification for regulating the fate of proteins and it is widely involved in plant development and stress responses. The SUMO molecules are conjugated to the lysine residues of substrate proteins via isopeptide bonds by enzyme reaction. SUMOylation is mediated by an enzyme cascade composed of a SUMO activating enzyme complex (E1), a SUMO-conjugating enzyme (E2) and usually a SUMO ligase (E3). Here, we report an efficient in vitro detection system for SUMOylation of plant proteins. We established a system for SUMOylation detection of plant proteins by reconstructing the Arabidopsis SUMOylation enzyme cascade in Escherichia coli. Using this system, SUMOylation of several substrates were detected via immunoblotting. Therefore, this system simplifies the SUMOylation detection of plant protein substrates and provides a powerful tool for functional analysis of SUMOylation in plant cells.

Proximity labeling (PL), a recently developed technique to detect protein-protein interactions and subcellular structural proteomes in living cells, has been successfully applied in various animal and plant systems. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity to a protein of interest (bait protein). With the catalysis of the enzyme, small molecular substrates such as biotin are covalently linked to endogenous proximal proteins, which can be further enriched and analyzed to identify the interactome of the bait protein. TurboID, a biotin ligase produced by directed evolution, has the advantages of non-toxicity and high catalytic efficiency. Using TurboID-based proximity labeling to analyze proximal proteins of bait proteins, we can study transient or weak protein interactions, which helps to understand the complex biological processes occurring inside cells. Here, we describe methods and related tips for TurboID-based proximal labeling in Arabidopsis thaliana, and hope to provide a reference for studying plant protein-protein interactions.

The plant circadian clock is a time-keeping molecular system, with a cycle of ~ 24 h. It was evolved to adapt to the diel rhythmic environmental cues generated by the self-rotation of the Earth. In addition to the time-keeping function, the circadian clock also regulates a plethora of plant growth and development processes by synchronizing the endogenous energy and metabolomic status. By sensing and integrating the dynamics of external environmental cues, circadian clock can coordinate gene expression at multiple levels, thus to increase the fitness of plants. Recently, there is an increasingly demand for measuring and assessing circadian phenotype for many non-circadian research field. Here, we summarized the currently available methods for detecting circadian rhythm, and show one most commonly used standard procedure for evaluating circadian phenotype in plants, which may help to provide the applicable technical assistance for the study of the circadian clock.