L. Andrew Staehelin

The field of plant cell biology has a rich history of discovery, going back to Robert Hooke’s discovery of cells themselves. The development of microscopes and preparation techniques has allowed for the visualization of subcellular structures, and the use of protein biochemistry, genetics, and molecular biology has enabled the identification of proteins and mechanisms that regulate key cellular processes. In this review, seven senior plant cell biologists reflect on the development of this research field in the past decades, including the foundational contributions that their teams have made to our rich, current insights into cell biology. Topics covered include signaling and cell morphogenesis, membrane trafficking, cytokinesis, cytoskeletal regulation, and cell wall biology. In addition, these scientists illustrate the pathways to discovery in this exciting research field.

Microscopic studies of chloroplasts can be traced back to the year 1678 when Antonie van Leeuwenhoek reported to the Royal Society in London that he saw green globules in grass leaf cells with his single-lens microscope. Since then, microscopic studies have continued to contribute critical insights into the complex architecture of chloroplast membranes and how their structure relates to function. This review is organized into three chronological sections: During the classic light microscope period (1678–1940), the development of improved microscopes led to the identification of green grana, a colorless stroma, and a membrane envelope. More recent (1990–2020) chloroplast dynamic studies have benefited from laser confocal and 3D-structured illumination microscopy. The development of the transmission electron microscope (1940–2000) and thin sectioning techniques demonstrated that grana consist of stacks of closely appressed grana thylakoids interconnected by non-appressed stroma thylakoids. When the stroma thylakoids were shown to spiral around the grana stacks as multiple right-handed helices, it was confirmed that the membranes of a chloroplast are all interconnected. Freeze-fracture and freeze-etch methods verified the helical nature of the stroma thylakoids, while also providing precise information on how the electron transport chain and ATP synthase complexes are non-randomly distributed between grana and stroma membrane regions. The last section (2000–2020) focuses on the most recent discoveries made possible by atomic force microscopy of hydrated membranes, and electron tomography and cryo-electron tomography of cryofixed thylakoids. These investigations have provided novel insights into thylakoid architecture and plastoglobules (summarized in a new thylakoid model), while also producing molecular-scale views of grana and stroma thylakoids in which individual functional complexes can be identified.

The insect-trapping leaves of Dionaea muscipula provide a model for studying the secretory pathway of an inducible plant secretory system. The leaf glands were induced with bovine serum albumin to secrete proteases that were characterized via zymogram activity gels over a 6-day period. The accompanying morphological changes of the endoplasmic reticulum (ER) and Golgi were analyzed using 3D electron tomography of glands preserved by high-pressure freezing/freeze substitution methods. Secretion of multiple cysteine and aspartic proteases occurred biphasically. The majority of the Golgi was organized in clusters consisting of 3–6 stacks surrounded by a cage-like system of ER cisternae. In these clusters, all Golgi stacks were oriented with their cis-most C1 cisterna facing an ER export site. The C1 Golgi cisternae varied in size and shape consistent with the hypothesis that they form de novo. Following induction, the number of ER-bound polysomes doubled, but no increase in COPII vesicles was observed. Golgi changes included a reduction in the number of cisternae per stack and a doubling of cisternal volume without increased surface area. Polysaccharide molecules that form the sticky slime cause swelling of the trans and trans Golgi network (TGN) cisternae. Peeling of the trans-most cisternae gives rise to free TGN cisternae. One day after gland stimulation, the free TGNs were frequently associated with loose groups of oriented actin-like filaments which were not seen in any other samples. These findings suggest that the secretory apparatus of resting gland cells is “overbuilt” to enable the cells to rapidly up-regulate lytic enzyme production and secretion in response to prey trapping.

Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression analyses of chloroplast proteins. Our study identified a linear developmental sequence with five assembly stages: tubulo-vesicular prothylakoids (24 h after imbibition [HAI]), sheet-like pregranal thylakoids that develop from the prothylakoids (36 HAI), proliferation of pro-grana stacks with wide tubular connections to the originating pregrana thylakoids (60 HAI), structural differentiation of pro-grana stacks and expanded stroma thylakoids (84 HAI), and conversion of the pro-grana stacks into mature grana stacks (120 HAI). Development of the planar pregranal thylakoids and the pro-grana membrane stacks coincides with the appearance of thylakoid-bound polysomes and photosystem II complex subunits at 36 HAI. ATP synthase, cytochrome b₆f, and light-harvesting complex II proteins are detected at 60 HAI, while PSI proteins and the curvature-inducing CURT1A protein appear at 84 HAI. If stromal ribosome biogenesis is delayed, prothylakoids accumulate until stromal ribosomes are produced, and grana-forming thylakoids develop after polysomes bind to the thylakoid membranes. In fzo-like (fzl) mutants, in which thylakoid organization is perturbed, pro-grana stacks in cotyledons form discrete, spiral membrane compartments instead of organelle-wide membrane networks, suggesting that FZL is involved in fusing membrane compartments together. Our data demonstrate that the assembly of thylakoid protein complexes, CURT1 proteins, and FZL proteins mediate distinct and critical steps in thylakoid biogenesis.

The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.

The Golgi apparatus contains multiple classes of cisternae that differ in structure, composition, and function, but there is no consensus about the number and definition of these classes. A useful way to classify Golgi cisternae is according to the trafficking pathways by which the cisternae import and export components. By this criterion, we propose that Golgi cisternae can be divided into three classes that correspond to functional stages of maturation. First, cisternae at the cisternal assembly stage receive COPII vesicles from the ER and recycle components to the ER in COPI vesicles. At this stage, new cisternae are generated. Second, cisternae at the carbohydrate synthesis stage exchange material with one another via COPI vesicles. At this stage, most of the glycosylation and polysaccharide synthesis reactions occur. Third, cisternae at the carrier formation stage produce clathrin-coated vesicles and exchange material with endosomes. At this stage, biosynthetic cargo proteins are packaged into various transport carriers, and the cisternae ultimately disassemble. Discrete transitions occur as a cisterna matures from one stage to the next. Within each stage, the structure and composition of a cisterna can evolve, but the trafficking pathways remain unchanged. This model offers a unified framework for understanding the properties of the Golgi in diverse organisms.

The cisternal progression/maturation model of Golgi trafficking predicts that cis‐Golgi cisternae are formed de novo on the cis‐side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high‐pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus flytrap) as determined by electron microscopy, electron tomography and immuno‐electron microscopy techniques. Our findings are as follows: (i) The cis‐most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3–5 COPII vesicles in contact with a C2 cis cisterna. (ii) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (iii) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (iv) C2‐Cn cis cisternae grow through COPII vesicle fusion. (v) ER‐resident proteins are recycled from cis cisternae to the ER via COPIa‐type vesicles. (vi) In S. dubia the C2 cisternae are capable of mediating the self‐assembly of scale protein complexes. (vii) In plants, ∼90% of native α‐mannosidase I localizes to medial Golgi cisternae. (viii) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre‐cis Golgi cisterna layer in mammalian cells.

We have investigated the structural events associated with vacuole biogenesis in root tip cells of tobacco (Nicotiana tabacum) seedlings preserved by high-pressure freezing and freeze-substitution techniques. Our micrographs demonstrate that the lytic vacuoles (LVs) of root tip cells are derived from protein storage vacuoles (PSVs) by cell type-specific sets of transformation events. Analysis of the vacuole transformation pathways has been aided by the phytin-dependent black osmium staining of PSV luminal contents. In epidermal and outer cortex cells, the central LVs are formed by a process involving PSV fusion, storage protein degradation, and the gradual replacement of the PSV marker protein α-tonoplast intrinsic protein (TIP) with the LV marker protein γ-TIP. In contrast, in the inner cortex and vascular cylinder cells, the transformation events are more complex. During mobilization of the stored molecules, the PSV membranes collapse osmotically upon themselves, thereby squeezing the vacuolar contents into the remaining bulging vacuolar regions. The collapsed PSV membranes then differentiate into two domains: (1) vacuole “reinflation” domains that produce pre-LVs, and (2) multilamellar autophagosomal domains that are later engulfed by the pre-LVs. The multilamellar autophagosomal domains appear to originate from concentric sheets of PSV membranes that create compartments within which the cytoplasm begins to break down. Engulfment of the multilamellar autophagic vacuoles by the pre-LVs gives rise to the mature LVs. During pre-LV formation, the PSV marker α-TIP disappears and is replaced by the LV marker γ-TIP. These findings demonstrate that the central LVs of root cells arise from PSVs via cell type-specific transformation pathways.