The joint analysis of the genome, epigenome, transcriptome, proteome and/or metabolome from single cells is transforming our understanding of cell biology in health and disease. In less than a decade, the field has seen tremendous technological revolutions that enable crucial new insights into the interplay between intracellular and intercellular molecular mechanisms that govern development, physiology and pathogenesis. In this Review, we highlight advances in the fast-developing field of single-cell and spatial multi-omics technologies (also known as multimodal omics approaches), and the computational strategies needed to integrate information across these molecular layers. We demonstrate their impact on fundamental cell biology and translational research, discuss current challenges and provide an outlook to the future.

Plant stress responses involve numerous changes at the molecular and cellular level and are regulated by highly complex signaling pathways. Studying protein-protein interactions (PPIs) and the resulting networks is therefore becoming increasingly important in understanding these responses. Crucial in PPI networks are the so-called hubs or hub proteins, commonly defined as the most highly connected central proteins in scale-free PPI networks. However, despite their importance, a growing amount of confusion and controversy seems to exist regarding hub protein identification, characterization and classification. In order to highlight these inconsistencies and stimulate further clarification, this review critically analyses the current knowledge on hub proteins in the plant interactome field. We focus on current hub protein definitions, including the properties generally seen as hub-defining, and the challenges and approaches associated with hub protein identification. Furthermore, we give an overview of the most important large-scale plant PPI studies of the last decade that identified hub proteins, pointing out the lack of overlap between different studies. As such, it appears that although major advances are being made in the plant interactome field, defining hub proteins is still heavily dependent on the quality, origin and interpretation of the acquired PPI data. Nevertheless, many hub proteins seem to have a reported role in the plant stress response, including transcription factors, protein kinases and phosphatases, ubiquitin proteasome system related proteins, (co-)chaperones and redox signaling proteins. A significant number of identified plant stress hubs are however still functionally uncharacterized, making them interesting targets for future research. This review clearly shows the ongoing improvements in the plant interactome field but also calls attention to the need for a more comprehensive and precise identification of hub proteins, allowing a more efficient systems biology driven unraveling of complex processes, including those involved in stress responses.

Expansion microscopy (ExM) enables nanoscale imaging of ribonucleic acids (RNA) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents which allow labeling and grafting of DNA oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation in situ hybridization chain reaction RNA-FISH techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multi-color staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ~50 nm in ×10 ExM, both for pre-and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable rapid functionalization of DNA oligonucleotides for expansion microscopy.