Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress.

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis (Arabidopsis thaliana) leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant trigalactosyldiacylglycerol1-1 sugar dependent 1-4 (tgd1-1 sdp1-4) that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we showed that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress.

SEED LIPID DROPLET PROTEIN1, SEED LIPID DROPLET PROTEIN2, and LIPID DROPLET PLASMA MEMBRANE ADAPTOR mediate lipid droplet-plasma membrane tethering.

Membrane contact sites (MCSs) are interorganellar connections that allow for the direct exchange of molecules, such as lipids or Ca2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here, we identified and characterized three proteins of Arabidopsis thaliana that form a lipid droplet (LD)-plasma membrane (PM) tethering complex in plant cells, namely LD-localized SEED LD PROTEIN (SLDP) 1 and SLDP2 and PM-localized LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein-protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and SLDP2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA is sufficient to reconstitute LD-PM tethering in Nicotiana tabacum pollen tubes, a cell type characterized by dynamically moving LDs in the cytosolic streaming. Furthermore, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD-PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis.

LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate.

Xylem sap is the major transport route for nutrients from roots to shoots. In the present study, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap and the growth of the xylem endophyte Brennaria salicis, and we also report transcriptional re-wiring of leaf defenses in poplar (Populus × canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. Approximately 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, whereas nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased, whereas salicylic acid and jasmonoyl-isoleucine decreased, with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 124 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N, except coumarins. Brennaria salicis growth was reduced in nutrient-supplemented xylem sap of low- and high- NO3- -fed plants compared to that of NH4+ -fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defenses related to systemic acquired and induced systemic resistance. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

Heat stress leads to rapid lipid remodeling and transcriptional adaptations in Nicotiana tabacum pollen tubes.

After reaching the stigma, pollen grains germinate and form a pollen tube that transports the sperm cells to the ovule. Due to selection pressure between pollen tubes, pollen grains likely evolved mechanisms to quickly adapt to temperature changes to sustain elongation at the highest possible rate. We investigated these adaptions in tobacco (Nicotiana tabacum) pollen tubes grown in vitro under 22°C and 37°C by a multi-omics approach including lipidomic, metabolomic, and transcriptomic analysis. Both glycerophospholipids and galactoglycerolipids increased in saturated acyl chains under heat stress (HS), while triacylglycerols (TGs) changed less in respect to desaturation but increased in abundance. Free sterol composition was altered, and sterol ester levels decreased. The levels of sterylglycosides and several sphingolipid classes and species were augmented. Most amino acid levels increased during HS, including the noncodogenic amino acids γ-amino butyrate and pipecolate. Furthermore, the sugars sedoheptulose and sucrose showed higher levels. Also, the transcriptome underwent pronounced changes with 1,570 of 24,013 genes being differentially upregulated and 813 being downregulated. Transcripts coding for heat shock proteins and many transcriptional regulators were most strongly upregulated but also transcripts that have so far not been linked to HS. Transcripts involved in TG synthesis increased, while the modulation of acyl chain desaturation seemed not to be transcriptionally controlled, indicating other means of regulation. In conclusion, we show that tobacco pollen tubes are able to rapidly remodel their lipidome under HS likely by post-transcriptional and/or post-translational regulation.

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea.

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

SEIPIN Isoforms Interact with the Membrane-Tethering Protein VAP27-1 for Lipid Droplet Formation.

SEIPIN proteins are localized to endoplasmic reticulum (ER)-lipid droplet (LD) junctions where they mediate the directional formation of LDs into the cytoplasm in eukaryotic cells. Unlike in animal and yeast cells, which have single SEIPIN genes, plants have three distinct SEIPIN isoforms encoded by separate genes. The mechanism of SEIPIN action remains poorly understood, and here we demonstrate that part of the function of two SEIPIN isoforms in Arabidopsis (Arabidopsis thaliana), AtSEIPIN2 and AtSEIPIN3, may depend on their interaction with the vesicle-associated membrane protein (VAMP)-associated protein (VAP) family member AtVAP27-1. VAPs have well-established roles in the formation of membrane contact sites and lipid transfer between the ER and other organelles, and here, we used a combination of biochemical, cell biology, and genetics approaches to show that AtVAP27-1 interacts with the N termini of AtSEIPIN2 and AtSEIPIN3 and likely supports the normal formation of LDs. This insight indicates that the ER membrane tethering machinery in plant cells could play a role with select SEIPIN isoforms in LD biogenesis at the ER, and additional experimental evidence in Saccharomyces cerevisiae supports the possibility that this interaction may be important in other eukaryotic systems.

Lipid droplets in plants and algae: Distribution, formation, turnover and function.

Plant oils represent an energy-rich and carbon-dense group of hydrophobic compounds. These oils are not only of economic interest, but also play important, fundamental roles in plant and algal growth and development. The subcellular storage compartments of plant lipids, referred to as lipid droplets (LDs), have long been considered relatively inert oil vessels. However, research in the last decade has revealed that LDs play far more dynamic roles in plant biology than previously appreciated, including transient neutral lipid storage, membrane remodeling, lipid signaling, and stress responses. Here we discuss recent developments in the understanding of LD formation, turnover and function in land plants and algae.

The evolution of the phenylpropanoid pathway entailed pronounced radiations and divergences of enzyme families.

Land plants constantly respond to fluctuations in their environment. Part of their response is the production of a diverse repertoire of specialized metabolites. One of the foremost sources for metabolites relevant to environmental responses is the phenylpropanoid pathway, which was long thought to be a land-plant-specific adaptation shaped by selective forces in the terrestrial habitat. Recent data have, however, revealed that streptophyte algae, the algal relatives of land plants, have candidates for the genetic toolkit for phenylpropanoid biosynthesis and produce phenylpropanoid-derived metabolites. Using phylogenetic and sequence analyses, we here show that the enzyme families that orchestrate pivotal steps in phenylpropanoid biosynthesis have independently undergone pronounced radiations and divergence in multiple lineages of major groups of land plants; sister to many of these radiated gene families are streptophyte algal candidates for these enzymes. These radiations suggest a high evolutionary versatility in the enzyme families involved in the phenylpropanoid-derived metabolism across embryophytes. We suggest that this versatility likely translates into functional divergence, and may explain the key to one of the defining traits of embryophytes: a rich specialized metabolism.

Finding new friends and revisiting old ones - how plant lipid droplets connect with other subcellular structures.

The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well-known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD-endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD-peroxisome connections, that facilitate the degradation of LD-stored triacylglycerols (TAGs), and the more recently discovered LD-plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s).

DIACYLGLYCEROL KINASE 5 regulates polar tip growth of tobacco pollen tubes.

Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear. We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant. Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes. We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.

Sphingolipid long-chain base hydroxylation influences plant growth and callose deposition in Physcomitrium patens.

Sphingolipids are enriched in microdomains in the plant plasma membrane (PM). Hydroxyl groups in the characteristic long-chain base (LCB) moiety might be essential for the interaction between sphingolipids and sterols during microdomain formation. Investigating LCB hydroxylase mutants in Physcomitrium patens might therefore reveal the role of certain plant sphingolipids in the formation of PM subdomains. Physcomitrium patens mutants for the LCB C-4 hydroxylase S4H were generated by homologous recombination. Plants were characterised by analysing their sphingolipid and steryl glycoside (SG) profiles and by investigating different gametophyte stages. s4h mutants lost the hydroxyl group at the C-4 position of their LCB moiety. Loss of this hydroxyl group caused global changes in the moss sphingolipidome and in SG composition. Changes in membrane lipid composition may trigger growth defects by interfering with the localisation of membrane-associated proteins that are crucial for growth processes such as signalling receptors or callose-modifying enzymes. Loss of LCB-C4 hydroxylation substantially changes the P. patens sphingolipidome and reveals a key role for S4H during development of nonvascular plants. Physcomitrium patens is a valuable model for studying the diversification of plant sphingolipids. The simple anatomy of P. patens facilitates visualisation of physiological processes in biological membranes.

Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings.

The developmental program of seed formation, germination, and early seedling growth requires not only tight regulation of cell division and metabolism, but also concerted control of the structure and function of organelles, which relies on specific changes in their protein composition. Of particular interest is the switch from heterotrophic to photoautotrophic seedling growth, for which cytoplasmic lipid droplets (LDs) play a critical role as depots for energy-rich storage lipids. Here, we present the results of a bottom-up proteomics study analyzing the total protein fractions and LD-enriched fractions in eight different developmental phases during silique (seed) development, seed germination, and seedling establishment in Arabidopsis (Arabidopsis thaliana). The quantitative analysis of the LD proteome using LD-enrichment factors led to the identification of six previously unidentified and comparably low-abundance LD proteins, each of which was confirmed by intracellular localization studies with fluorescent protein fusions. In addition to these advances in LD protein discovery and the potential insights provided to as yet unexplored aspects in plant LD functions, our data set allowed for a comparative analysis of the LD protein composition throughout the various developmental phases examined. Among the most notable of the alterations in the LD proteome were those during seedling establishment, indicating a switch in the physiological function(s) of LDs after greening of the cotyledons. This work highlights LDs as dynamic organelles with functions beyond lipid storage.

PUX10 Is a Lipid Droplet-Localized Scaffold Protein That Interacts with CELL DIVISION CYCLE48 and Is Involved in the Degradation of Lipid Droplet Proteins.

The number of known proteins associated with plant lipid droplets (LDs) is small compared with other organelles. Many aspects of LD biosynthesis and degradation are unknown, and identifying and characterizing candidate LD proteins could help elucidate these processes. Here, we analyzed the proteome of LD-enriched fractions isolated from tobacco (Nicotiana tabacum) pollen tubes. Proteins that were highly enriched in comparison with the total or cytosolic fraction were further tested for LD localization via transient expression in pollen tubes. One of these proteins, PLANT UBX DOMAIN-CONTAINING PROTEIN10 (PUX10), is a member of the plant UBX domain-containing (PUX) protein family. This protein localizes to LDs via a unique hydrophobic polypeptide sequence and can recruit the AAA-type ATPase CELL DIVISION CYCLE48 (CDC48) protein via its UBX domain. PUX10 is conserved in Arabidopsis thaliana and expressed in embryos, pollen tubes, and seedlings. In pux10 knockout mutants in Arabidopsis, LD size is significantly increased. Proteomic analysis of pux10 mutants revealed a delayed degradation of known LD proteins, some of which possessed ubiquitination sites. We propose that PUX10 is involved in a protein degradation pathway at LDs, mediating an interaction between polyubiquitinated proteins targeted for degradation and downstream effectors such as CDC48.

Large-scale reduction of the Bacillus subtilis genome: consequences for the transcriptional network, resource allocation, and metabolism.

Understanding cellular life requires a comprehensive knowledge of the essential cellular functions, the components involved, and their interactions. Minimized genomes are an important tool to gain this knowledge. We have constructed strains of the model bacterium, Bacillus subtilis, whose genomes have been reduced by ∼36%. These strains are fully viable, and their growth rates in complex medium are comparable to those of wild type strains. An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions affect the transcription regulatory network of the cell, translation resource allocation, and metabolism. A comparison of gene counts and resource allocation demonstrates drastic differences in the two parameters, with 50% of the genes using as little as 10% of translation capacity, whereas the 6% essential genes require 57% of the translation resources. Taken together, the results are a valuable resource on gene dispensability in B. subtilis, and they suggest the roads to further genome reduction to approach the final aim of a minimal cell in which all functions are understood.

Identification of the first glyphosate transporter by genomic adaptation.

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase.

Dynamics of the Pollen Sequestrome Defined by Subcellular Coupled Omics.

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.