Biomolecular condensates are triggered by multivalent interactions conferred by the intrinsically disordered regions and/or interacting domains within the constituents. While light microscopy has provided powerful tools to study the dynamics of intracellular condensates, electron microscopy (EM) gives more detailed insights into their ultrastructure and spatial connectivity with membrane system. In this chapter, we describe the methods for detecting the membraneless condensates in plant cells by high-pressure freezing -based EM coupled with immuno-gold labeling and correlative light electron microscopy techniques, which may benefit researchers in future studies.

Macroautophagy, hereafter autophagy, plays a crucial role in the degradation of harmful or unwanted cellular components through a double-membrane autophagosome. Upon autophagosome fusion with the vacuole, the degraded materials are subsequently recycled to generate macromolecules, contributing to cellular homeostasis, metabolism, and stress tolerance in plants. A hallmark during autophagy is the formation of isolation membrane structure named as phagophore, which undergoes multiple steps to become as a complete double-membrane autophagosome. Methodologies have been developed in recent years to observe and quantify the autophagic process, which greatly advance knowledge of autophagosome biogenesis in plant cells. In this chapter, we will introduce two methods to dissect the autophagosome-related structures in the Arabidopsis plant cells, including the correlative light and electron microscopy, to map the ultrastructural feature of autophagosomal structures, and time-lapse imaging to monitor the temporal recruitment of autophagy machinery during autophagosome formation.

A working pipeline for proteomic analysis of secreted vesicle proteins from the plant cells has been developed using urea and mass spectrometry-compatible detergent RapiGest SF, where vesicles could be efficiently lysed and membrane-bound proteins could be efficiently dissolved and digested. The vesicle lysis and the protein digestion procedures are performed within one tube to minimize the protein loss. The protein digest is analyzed using LC-MS/MS after desalting with an SPE spin column.

Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved from yeast to mammals, both conventional and unconventional protein secretion pathways have been revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide are translocated into the endoplasmic reticulum and transported to the extracellular region via the endomembrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated findings and provide a comprehensive overview of protein secretion pathways in plants.

We present a new set of computational tools that enable accurate and widely applicable 3D segmentation of nuclei in various 3D digital organs. We have developed an approach for ground truth generation and iterative training of 3D nuclear segmentation models, which we applied to popular CellPose, PlantSeg and StarDist algorithms. We provide two high-quality models trained on plant nuclei that enable 3D segmentation of nuclei in datasets obtained from fixed or live samples, acquired from different plant and animal tissues, and stained with various nuclear stains or fluorescent protein-based nuclear reporters. We also share a diverse high-quality training dataset of about 10,000 nuclei. Furthermore, we advanced the MorphoGraphX analysis and visualization software by, among other things, providing a method for linking 3D segmented nuclei to their surrounding cells in 3D digital organs. We found that the nuclear-to-cell volume ratio varies between different ovule tissues and during the development of a tissue. Finally, we extended the PlantSeg 3D segmentation pipeline with a proofreading tool that uses 3D segmented nuclei as seeds to correct cell segmentation errors in difficult-to-segment tissues.

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The engineering of plant cell cultures to produce high-value natural products is suggested to be a safe, low-cost, and environmentally friendly route to produce a wide range of chemicals. Given that the expression of heterologous biosynthetic pathways in plant tissue culture is limited by a lack of detailed protocols, the biosynthesis of high-value metabolites in plant cell culture is constrained compared with that in microbes. However, both Arabidopsis thaliana and Nicotiana benthamiana can be efficiently transformed with multigene constructs to produce high-value natural products in stable plant cell cultures. This chapter provides a detailed protocol as to how to engineer the plant cell culture as bio-factories for metabolite biosynthesis.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

Statistics and experimental design are important tools for plant cell and tissue culture researchers and should be used when planning and conducting experiments as well as during the analysis and interpretation of experimental results. The chapter provides basic concepts important to the statistical analysis of data obtained from plant tissue culture experiments and illustrates the application of common statistical procedures to analyze binomial, count, and continuous data for experiments with different treatment factors as well as identifying trends of dosage treatment factors.

Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.