Cryo-electron tomography (cryo-ET) is a transformative technique in cell biology that enables three-dimensional visualization of cellular structures in near-native states and at nanometer and even subnanometer resolution. Unlike traditional imaging methods, cryo-ET preserves the ultrastructure of cells without chemical fixation or staining, allowing researchers to observe macromolecular complexes in situ. Cryo-focused ion beam milling has overcome sample thickness limitations, enabling high-resolution imaging of complex and large specimens. When combined with correlative light microscopy and subtomogram averaging, cryo-ET can localize and resolve macromolecular assemblies within the cell. We discuss how cryo-ET has provided unprecedented insights into cellular architecture by bridging the gap between molecular and cellular scales and highlight examples in photosynthetic organisms. We also discuss new efforts to increase automation, throughput, and validation that make cryo-ET accessible to a larger community of scientists, including plant biologists.

Microautophagy (MI-autophagy) is an umbrella term for intracellular degradative pathways that entail the invagination or protrusion of the limiting membranes of endolysosomal compartments, that is, late endosomes and mammalian lysosomes or yeast and plant vacuoles, followed by pinching-off of the membrane into the lumen of the organelle. During these processes, the material specifically and nonspecifically targeted for degradation is sequestered within the invaginating or protuberating membrane. In contrast to macroautophagy, the molecular mechanisms underlying MI-autophagy are largely unknown due to their diversity and complexity in location, regulation and molecular machinery requirements. Here, we review recent progress in the field of MI-autophagy, describing the molecular basis and functions of the MI-autophagic pathways reported to date in eukaryotic cells, from yeast to mammalian and plant cells.

The nuclear pore complex controls the movement of proteins into and out of the nucleus, allowing cells to regulate protein localization and abundance. This process influences how organisms respond to environmental stimuli. Components of the nuclear pore complex, including the NUP107-160 sub-complex, NUP133, NUP85, and NENA, are required for root nodulation and arbuscular mycorrhization in Lotus japonicus. However, the specific role of these nucleoporins in symbiotic signaling was poorly understood. Through reverse genetics, we discovered that NUP133 is also required for symbiosis in Medicago truncatula, although the mutant phenotypes were less pronounced than in Lotus. Overexpression of the symbiotic ion channels Medicago DMI1 and Lotus Castor and Pollux in the Lotus Ljnup133, Ljnup85, and Ljnena mutants partially alleviated the nodulation defects. Notably, in NUP107-160 sub-complex mutants of Lotus and Medicago, the accumulation of GFP-labeled Pollux and DMI1 on the inner nuclear membrane was reduced, indicating the NUP107-160 sub-complex plays a key role in regulating the distribution of DMI1 and Pollux on the nuclear envelope. This highlights the extreme sensitivity of nodulation in Lotus to changes in the abundance of Pollux on the inner nuclear membrane. In contrast, Medicago appears to exhibit greater tolerance to alterations in the distribution of DMI1 on the nuclear envelope.

Precursor mRNA (pre-mRNA) splicing occurs co-transcriptionally and is coupled to transcription through the coordinated assembly of the splicing and transcription machinery. Splicing factor 3B subunit 2 (SF3B2) plays a critical role in pre-mRNA splicing and facilitates spliceosome assembly in humans, but its function in plants remains unclear. Here, we demonstrate that the Arabidopsis thaliana SF3B2 homolog AtSF3B2 interacts with the splicing factors AtU2AF65B and AtSYF2 via its conserved DUF382 domain. As a negative regulator of floral transition, AtSF3B2 binds to pre-mRNAs and modulates the splicing of its target genes, including the central floral repressor FLOWERING LOCUS C (FLC), its antisense transcript COOLAIR, and their regulator WRKY63. Furthermore, AtSF3B2 promotes the transcription of these genes by interacting with the RNA polymerase II (Pol II) subunit NRPB12 and by binding directly to DNA to influence Pol II enrichment. RNA sequencing analyses reveal that the AtSF3B2 mutation predominantly results in intron retention and exon skipping, especially for shorter exons with a lower GC content. A subset of flowering regulators, including FLM, was identified as an AtSF3B2 target. Additionally, AtSF3B2 functions in high temperature-dependent flowering by modulating transcription and splicing of FLM. Together, our findings reveal transcriptional and post-transcriptional roles for AtSF3B2 in the flowering transition.

Post-Golgi trafficking in plants regulates transport to and from the cell surface, vacuolar trafficking, and recycling pathways within the endomembrane system. Endosomes serve as central hubs in these pathways, managing the composition of lipids and proteins in the plasma membrane and vacuole in response to developmental signals or environmental changes. Once internalized via endocytosis, plasma membrane proteins are directed to the trans-Golgi Network (TGN), which acts as an early endosome. From the TGN, proteins can either be sent back to the plasma membrane or trafficked to multivesicular endosomes (MVEs) for further sorting and ultimate delivery to the vacuole for degradation. Key molecular assemblies such as the retromer, the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, small GTPases, adaptor proteins, and SNAREs associate with distinct domains of endosomal membranes to facilitate protein sorting and membrane remodeling. This review focuses on the roles of endosomes in post-Golgi trafficking, mechanisms of cargo sorting, and membrane remodeling.

The pollen tube is a remarkable cellular structure that carries the sperm cells to the ovules for fertilization.It derives from the vegetative cell within the pollen grain,and it needs to grow very quickly through the female floral tissues to outcompete other pollen tubes in the fertilization process.This is achieved by active vesicle trafficking at the pollen tube distal zone through a highly polarized growth me-chanism.The apical zone,often called the clear zone,is located at the growing tip of the pollen tube.

Endocytosis and endosomal trafficking of plasma membrane proteins for degradation regulate cellular homeostasis and development. As part of these processes, ubiquitinated plasma membrane proteins (cargo) are recognized, clustered, and sorted into intraluminal vesicles of multivesicular endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. At endosomes, ESCRT proteins recognize ubiquitinated cargo and mediate the deformation of the endosomal membrane in a negative geometry, away from the cytosol. ESCRTs are organized in five major complexes that are sequentially recruited to the endosomal membrane where they mediate its vesiculation and cargo sequestration. ESCRTs also participate in other membrane remodeling events and are widely conserved across organisms, both eukaryotes and prokaryotes. Plants contain both conserved and unique ESCRT components and show a general trend toward gene family expansion. Plant endosomes show a wide range of membrane budding patterns with potential implications in cargo sequestration efficiency, plant development, and hormone signaling. Understanding the diversification and specialization of plant ESCRT proteins can provide valuable insights in the mechanisms of ESCRT-mediated membrane bending. In this review, we discuss the endosomal function of ESCRT proteins, their unique features in plants, and the potential connections to the modes of plant endosomal vesiculation.

Proteolysis is an essential cellular function mediating the processing and turnover of proteins to remove damaged or inactive proteins, alter protein function (binding or enzymatic activities), and ensure appropriate protein stoichiometries in the cell.The post-translational control of protein stability is also a central feature of cellular signaling in eukaryotes, i.e. regulating the turnover of important regulatory proteins, and is crucial for almost all aspects of plant biology, including vegetative growth, development, reproduction, and stress responses.Proteolysis plays a central role in hormone signaling pathways, plant defense against pests and pathogens, abiotic stress responses, and basic cell functions like the cell cycle, metabolism, organellar biogenesis and maintenance, and senescence.Compared with animals, plant genomes encode a highly expanded number of components related to proteolytic pathways including the ubiquitin-proteasome system, autophagy, programmed cell death, endosomal trafficking, and organelle-associated protein degradation.Recent advances in molecular genetics and cell biology, microscopy and high-resolution imaging, in vivo labeling, proteomics, mass spectrometry, and structural biology have led to new insights and understanding in many areas of plant proteolysis, including autophagy of chloroplasts and other organelles, degradation of membrane proteins, the discovery of plant N-degron pathways, and proteolytic processing involved in plant development and environmental responses (immunity and abiotic stress).Knowledge of plant proteolytic systems is also important for agriculture and plant breeding, given the impact on plant growth and development related to yield, as well as plant resistance to pests and diseases.This focus issue on plant proteolysis includes two letters to the editor, one commentary, nine review articles, and eight original research articles.The two letters address the question of whether the polyubiquitin pathway operates inside intact chloroplasts.The question is posed by van Wijk and Adam (2024), with a Reply from Jarvis et al. (2024).Researchers on both sides of the debate make valid points to be considered.We eagerly await more definitive data to arrive at a complete understanding of protein degradation inside chloroplasts.The commentary by Eckardt et al.

Multivesicular endosomes (MVEs) sequester membrane proteins destined for degradation within intralumenal vesicles (ILVs), a process mediated by the membrane-remodeling action of Endosomal Sorting Complex Required for Transport (ESCRT) proteins. In Arabidopsis , endosomal membrane constriction and scission are uncoupled, resulting in the formation of extensive concatenated ILV networks and enhancing cargo sequestration efficiency. Here, we used a combination of electron tomography, computer simulations, and mathematical modeling to address the questions of when concatenated ILV networks evolved in plants and what drives their formation. Through morphometric analyses of tomographic reconstructions of endosomes across yeast, algae, and various land plants, we have found that ILV concatenation is widespread within plant species, but only prevalent in seed plants, especially in flowering plants. Multiple budding sites that require the formation of pores in the limiting membrane were only identified in hornworts and seed plants, suggesting that this mechanism has evolved independently in both plant lineages. To identify the conditions under which these multiple budding sites can arise, we used particle-based molecular dynamics simulations and found that changes in ESCRT filament properties, such as filament curvature and membrane binding energy, can generate the membrane shapes observed in multiple budding sites. To understand the relationship between membrane budding activity and ILV network topology, we performed computational simulations and identified a set of membrane remodeling parameters that can recapitulate our tomographic datasets.

At the heart of all biological processes lies the control of nuclear gene expression, which is primarily achieved through the action of transcription factors (TFs) that generally contain a nuclear localization signal (NLS) to facilitate their transport into the nucleus. However, some TFs reside in the cytoplasm in a transcriptionally inactive state and only enter the nucleus in response to specific signals, which in plants include biotic or abiotic stresses. These extra-nuclear TFs can be found in the cytosol or associated with various membrane systems, including the endoplasmic reticulum and plasma membrane. They may be integral proteins with transmembrane domains or associate peripherally with the lipid bilayer via acylation or membrane-binding domains. Although over 30 plant TFs, most of them involved in stress responses, have been experimentally shown to reside outside the nucleus, computational predictions suggest that this number is much larger. Understanding how extra-nuclear TFs are trafficked into the nucleus is essential for reconstructing transcriptional regulatory networks that govern major cellular pathways in response to biotic and abiotic signals. Here, we provide a perspective on what is known on plant extranuclear-nuclear TF retention, nuclear trafficking, and the post-translational modifications that ultimately enable them to regulate gene expression upon entering the nucleus.

Autophagy is a process of cellular self-eating, which allows organisms to eliminate and recycle unwanted components and damaged organelles to maintain cellular homeostasis. It is an important process in the development of eukaryotic organisms. Autophagy plays a critical role in many physiological processes in plants such as nutrient remobilization, cell death, immunity, and abiotic stress responses. Autophagy thus represents an obvious target for generating resilient crops. During plant development, autophagy is also implicated in the differentiation and maturation of various cell types and plant organs, including root cap cells, tracheary elements, gametes, fruits and seeds. Here, we review our current understanding and recent advances of plant autophagy including insight into autophagy regulation and signaling as well as autophagosome membrane biogenesis. In addition, we describe how autophagy contributes to development, metabolism, biotic and abiotic stress tolerance and where the autophagic field is heading in terms of applied research for crop improvement.

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of the pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.

The selective turnover of macromolecules by autophagy provides a critical homeostatic mechanism for recycling cellular constituents and for removing superfluous and damaged organelles, membranes, and proteins. To better understand how autophagy impacts seed maturation and nutrient storage, we studied maize (Zea mays) endosperm in its early and middle developmental stages via an integrated multiomic approach using mutants impacting the core macroautophagy factor AUTOPHAGY (ATG)-12 required for autophagosome assembly. Surprisingly, the mutant endosperm in these developmental windows accumulated normal amounts of starch and Zein storage proteins. However, the tissue acquired a substantially altered metabolome, especially for compounds related to oxidative stress and sulfur metabolism, including increases in cystine, dehydroascorbate, cys-glutathione disulfide, glucarate, and galactarate, and decreases in peroxide and the antioxidant glutathione. While changes in the associated transcriptome were mild, the proteome was strongly altered in the atg12 endosperm, especially for increased levels of mitochondrial proteins without a concomitant increase in mRNA abundances. Although fewer mitochondria were seen cytologically, a heightened number appeared dysfunctional based on the accumulation of dilated cristae, consistent with attenuated mitophagy. Collectively, our results confirm that macroautophagy plays a minor role in the accumulation of starch and storage proteins during maize endosperm development but likely helps protect against oxidative stress and clears unneeded/dysfunctional mitochondria during tissue maturation.

Electron tomography (ET) imaging of high-pressure frozen/freeze-substituted samples provides a unique opportunity to study structural details of organelles and cytoskeletal arrays in plant cells. In this chapter, we discuss approaches for sample preparation by cryofixation at high pressure, freeze substitution, and resin embedding. We also include pipelines for data collection for electron tomography at ambient temperature, tomogram calculation, and segmentation.

There is a strong need to develop the knowledge base that will enable the rational design of improved crop species for sustainable agriculture and biomass production. To attain this goal, researchers must learn how plant cells integrate metabolism and cellular dynamics during growth and in response to environmental challenges. Meeting these goals require data sharing and the development and training of faculty, post-doctoral fellows, graduate and undergraduate students in state-of-the-art quantitative integrative cell biology methods. The inaugural Plant Cell Biology International (PCBI) meeting capitalized on the interaction between two well-established plant cell biology communities across the Atlantic, the Midwest Plant Cell Dynamics (PCD) and the European Network for Plant Endomembrane Research (ENPER) and provided a framework to meet these goals. The conference took place in person successfully during August 1-5th 2022 in Crete, Greece. The conference offered opportunities for undergraduate and graduate students, postdocs, young investigators across the globe to present their results and interact across different disciplines. The conference provided an open and inclusive forum for oral presentations that allowed all labs to share research results. It was attended over 80 % by students, postdocs and young investigators. Workshops on best practices for image acquisition and processing pipelines, publication guidelines by scientific journal editors, professional development and well-being benefited the participants at large. Overall, this was the inaugural joint Plant Cell Biology International conference that for the first time officially brought together plant cell biologists to discuss opportunities and challenges in the field to gain a mechanistic understanding of complex biological processes and advance sustainable agriculture. Due to its success, it was rescheduled for 2025 in Crete, to continue strengthening the plant cell biology community and exchanging knowledge and expertise across the globe.

"Chloroplast microautophagy: A green role for NBR1." Autophagy, ahead-of-print(ahead-of-print), pp. 1–2 AcknowledgementsThis work was supported, in part, by grants from the U.S. National Science Foundation IOS-1840687 and U.S. Department of Energy grant DE-SC0019013.Disclosure StatementNo potential conflict of interest was reported by the author(s).Additional informationFundingThe work was supported by the U.S. National Science Foundation [IOS-1840687]; U.S. Department of Energy [DE-SC0019013].

Plant tissues are often considered not ideal for fluorescence imaging because of the pervasive intrinsic fluorescence of many plant metabolites and the intricate interactions with light of the many semi-crystalline polymers at the cell wall. Our project aims to take advantage of this observed shortcoming by developing a label-free, optical microscopy platform for characterizing multiple fingerprints of important cell wall components and stress-related, at subcellular scale resolution. The new imaging system can collect fingerprints from both emitted and scattered light that can inform on the chemical nature, subcellular distribution, anisotropy, and molecular environment of multiple cell wall components in intact plant tissues. We are combining these imaging capabilities with computational tools that enable correlated registration, integration, and analysis. This fully integrated, multiparametric optical system will be used to address biological problems connected to cell wall assembly in grasses. This includes a focus on developmental and environmental variation of cell wall impregnation with silica, lignin, suberin, and cutin in different tissues and cell types. Our research plan comprises three main goals: (1) To develop an accessible imaging platform and associated open-source software able to extract and integrate fingerprints from fluorescence-associated (multispectral emission, lifetime, and polarization), wide-field polarimetry, second harmonic generation (SHG), and stimulated Raman scattering signals (SRS); (2) To determine unique combination of fingerprints for various cell wall components and selected metabolites; (3) To analyze the process of cell wall silicification in grasses and determine how silicification affects cell wall properties and lignin, cutin, and suberin deposition in other cell types under differ stress conditions.

The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.