Tomato Pollen Research Articles

Evaluation of Natural and Factitious Food Sources for Pronematus ubiquitus on Tomato Plants.

Simple SummaryBiocontrol practitioners have increasingly released generalist predators to control a variety of greenhouse pests. In this study, the effects of alternative food sources on the oviposition rate and the population dynamics of the mite Pronematus ubiquitus were assessed. The alternative food sources were selected among those the mite may encounter or be applied to the tomato crop.Pronematus ubiquitus (McGregor) is a small iolinid mite that is capable of establishing on tomato plants. Once established, this mite has been shown to control both tomato russet mite, Aculops lycopersici (Tryon) (Acari: Eriophyidae), and tomato powdery mildew (Oidium neolycopersici L. Kiss). In the present study, we explored the effects of a number of alternative food sources on the oviposition rate in the laboratory. First, we assessed the reproduction on food sources that P. ubiquitus can encounter on a tomato crop: tomato pollen and powdery mildew, along with tomato leaf and Typha angustifolia L. In a second laboratory experiment, we evaluated the oviposition rate on two prey mites: the astigmatid Carpoglyphus lactis L. (Acari: Carpoglyphidae) and the tarsonemid Tarsonemus fusarii Cooreman (Acari: Tarsonemidae). Powdery mildew and C. lactis did not support reproduction, whereas tomato pollen and T. fusarii did promote egg laying. However, T. angustifolia pollen resulted in a higher oviposition in both experiments. In a greenhouse trial on individual caged tomato plants, we evaluated the impact of pollen supplementation frequency on the establishment of P. ubiquitus. Here, a pollen addition frequency of every other week was required to allow populations of P. ubiquitus to establish.

Phytochrome interacting factor 3 regulates pollen mitotic division through auxin signalling and sugar metabolism pathways in tomato.

Summary The development of viable pollen determines male fertility, and is crucial for reproduction in flowering plants. Phytochrome interacting factor 3 (PIF3) acts as a central regulator of plant growth and development, but its relationship with pollen development has not been determined.Through genetic, histological and transcriptomic analyses, we identified an essential role for SlPIF3 in regulating tomato (Solanum lycopersicum) pollen development. Knocking out SlPIF3 using clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 resulted in pollen mitosis I arrest, and a failure to form viable pollen. We further demonstrated that both glutamate synthase 1 (SlGLT1) and cell wall invertase 9 (SlCWIN9), involved in auxin and sugar homeostasis, respectively, colocalised with SlPIF3 in the anthers and were directly regulated by SlPIF3. Knockout of either SlGLT1 or SlCWIN9 phenocopied the pollen phenotype of SlPIF3 knockout (Slpif3) lines. Slpif3 fertility was partially restored by exogenous auxin indole‐3‐acetic acid in a dose‐dependent manner.This study reveals a mechanism by which SlPIF3 regulates pollen development and highlights a new strategy for creating hormone‐regulated genic male sterile lines for tomato hybrid seed production.

1-Aminocyclopropane-1-carboxylic acid stimulates tomato pollen tube growth independently of ethylene receptors.

The plant hormone ethylene plays vital roles in plant development, including pollen tube (PT) growth. Many studies have used the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as a tool to trigger ethylene signaling. Several studies have suggested that ACC can act as a signal molecule independently of ethylene, inducing responses that are distinct from those induced by ethylene. In this study, we confirmed that ethylene receptor function is essential for promoting PT growth in tomato, but interestingly, we discovered that ACC itself can act as a signal that also promotes PT growth. Exogenous ACC stimulated PT growth even when ethylene perception was inhibited either chemically by treating with 1-methylcyclopropene (1-MCP) or genetically by using the ethylene-insensitive Never Ripe (NR) mutant. Treatment with aminoethoxyvinylglycine, which reduces endogenous ACC levels, led to a reduction of PT growth, even in the NR mutants. Furthermore, GUS activity driven by an EIN3 Binding Site promoter (EBS:GUS transgene) was triggered by ACC in the presence of 1-MCP. Taken together, these results suggest that ACC signaling can bypass the ethylene receptor step to stimulate PT growth and EBS driven gene expression.

Two Carbohydrate-Based Natural Extracts Stimulate in vitro Pollen Germination and Pollen Tube Growth of Tomato Under Cold Temperatures.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular markers implicated in pollen tube growth. Neither the number of callose plugs nor the CALLOSE SYNTHASE genes expression were significantly different between the control and the biostimulated pollen tubes when pollens were cultivated under cold conditions. PECTIN METHYLESTERASE (PME) activities were also similar but a basic PME isoform was not produced or inactive in pollen grown at 8°C. Nevertheless, NADPH oxidase (RBOH) gene expression was correlated with a higher number of viable pollen tubes in biostimulated pollen tubes compared to the control. Our results showed that the two carbohydrate-based products were able to reduce in vitro the effect of cold temperatures on tomato pollen tube growth and at least for one of them to modulate reactive oxygen species production.

Sequential Deposition and Remodeling of Cell Wall Polymers During Tomato Pollen Development.

The cell wall of a mature pollen grain is a highly specialized, multilayered structure. The outer, sporopollenin-based exine provides protection and support to the pollen grain, while the inner intine, composed primarily of cellulose, is important for pollen germination. The formation of the mature pollen grain wall takes place within the anther with contributions of cell wall material from both the developing pollen grain as well as the surrounding cells of the tapetum. The process of wall development is complex; multiple cell wall polymers are deposited, some transiently, in a controlled sequence of events. Tomato (Solanum lycopersicum) is an important agricultural crop, which requires successful fertilization for fruit production as do many other members of the Solanaceae family. Despite the importance of pollen development for tomato, little is known about the detailed pollen gain wall developmental process. Here, we describe the structure of the tomato pollen wall and establish a developmental timeline of its formation. Mature tomato pollen is released from the anther in a dehydrated state and is tricolpate, with three long apertures without overlaying exine from which the pollen tube may emerge. Using histology and immunostaining, we determined the order in which key cell wall polymers were deposited with respect to overall pollen and anther development. Pollen development began in young flower buds when the premeiotic microspore mother cells (MMCs) began losing their cellulose primary cell wall. Following meiosis, the still conjoined microspores progressed to the tetrad stage characterized by a temporary, thick callose wall. Breakdown of the callose wall released the individual early microspores. Exine deposition began with the secretion of the sporopollenin foot layer. At the late microspore stage, exine deposition was completed and the tapetum degenerated. The pollen underwent mitosis to produce bicellular pollen; at which point, intine formation began, continuing through to pollen maturation. The entire cell wall development process was also punctuated by dynamic changes in pectin composition, particularly changes in methyl-esterified and de-methyl-esterified homogalacturonan.

Sheet-like clay nanoparticles deliver RNA into developing pollen to efficiently silence a target gene.

Topical application of double-stranded RNA (dsRNA) can induce RNA interference (RNAi) and modify traits in plants without genetic modification. However, delivering dsRNA into plant cells remains challenging. Using developing tomato (Solanum lycopersicum) pollen as a model plant cell system, we demonstrate that layered double hydroxide (LDH) nanoparticles up to 50 nm in diameter are readily internalized, particularly by early bicellular pollen, in both energy-dependent and energy-independent manners and without physical or chemical aids. More importantly, these LDH nanoparticles efficiently deliver dsRNA into tomato pollen within 2-4 h of incubation, resulting in an 89% decrease in transgene reporter mRNA levels in early bicellular pollen 3-d post-treatment, compared with a 37% decrease induced by the same dose of naked dsRNA. The target gene silencing is dependent on the LDH particle size, the dsRNA dose, the LDH-dsRNA complexing ratio, and the treatment time. Our findings indicate that LDH nanoparticles are an effective nonviral vector for the effective delivery of dsRNA and other biomolecules into plant cells.

Ethylene signaling modulates tomato pollen tube growth through modifications of cell wall remodeling and calcium gradient.

Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and both ethylene production and perception seem critical at the time of fertilization. The full gene set regulated by ethylene during PT growth is unknown. To study this, we used various EThylene Receptor (ETR) tomato (Solanum lycopersicum) mutants: etr3-ko, a loss-of-function (LOF) mutant; and NR (NEVER RIPE), a gain-of-function (GOF) mutant. The etr3-ko PTs grew faster than wild-type (WT) PTs. Oppositely, NR PT elongation was slower than in WT, and PTs displayed larger diameters. ETR mutations result in feedback control of ethylene production. Furthermore, ethylene treatment of germinating pollen grains increased PT length in etr-ko mutants and WT, but not in NR. Treatment with the ethylene perception inhibitor 1-methylcyclopropene decreased PT length in etr-ko mutants and WT, but had no effect on NR. This confirmed that ethylene regulates PT growth. The comparison of PT transcriptomes in LOF and GOF mutants, etr3-ko and NR, both harboring mutations of the ETR3 gene, revealed that ethylene perception has major impacts on cell wall- and calcium-related genes as confirmed by microscopic observations showing a modified distribution of the methylesterified homogalacturonan pectic motif and of calcium load. Our results establish links between PT growth, ethylene, calcium, and cell wall metabolism, and also constitute a transcriptomic resource.

Ornithine decarboxylase genes contribute to S-RNase-independent pollen rejection.

Unilateral incompatibility (UI) manifests as pollen rejection in the pistil, typically when self-incompatible (SI) species are pollinated by self-compatible (SC) relatives. In the Solanaceae, UI occurs when pollen lack resistance to stylar S-RNases, but other, S-RNase-independent mechanisms exist. Pistils of the wild tomato Solanum pennellii LA0716 (SC) lack S-RNase yet reject cultivated tomato (Solanum lycopersicum, SC) pollen. In this cross, UI results from low pollen expression of a farnesyl pyrophosphate synthase gene (FPS2) in S. lycopersicum. Using pollen from fps2-/- loss-of-function mutants in S. pennellii, we identified a pistil factor locus, ui3.1, required for FPS2-based pollen rejection. We mapped ui3.1 to an interval containing 108 genes situated on the IL 3-3 introgression. This region includes a cluster of ornithine decarboxylase (ODC2) genes, with four copies in S. pennellii, versus one in S. lycopersicum. Expression of ODC2 transcript was 1,034-fold higher in S. pennellii than in S. lycopersicum styles. Pistils of odc2-/- knockout mutants in IL 3-3 or S. pennellii fail to reject fps2 pollen and abolish transmission ratio distortion (TRD) associated with FPS2. Pollen of S. lycopersicum express low levels of FPS2 and are compatible on IL 3-3 pistils, but incompatible on IL 12-3 × IL 3-3 hybrids, which express both ODC2 and ui12.1, a locus thought to encode the SI proteins HT-A and HT-B. TRD observed in F2 IL 12-3 × IL 3-3 points to additional ODC2-interacting pollen factors on both chromosomes. Thus, ODC2 genes contribute to S-RNase independent UI and interact genetically with ui12.1 to strengthen pollen rejection.

Find your identity – methylation dynamics in the sperm cell lineage

Find your identity – methylation dynamics in the sperm cell lineage

An Ethylene Over-Producing Mutant of Tomato (<i>Solanum lycopersicum</i>), Epinastic, Exhibits Tolerance to High Temperature Conditions

Above-optimal temperatures reduce yield in many crops, including tomato, largely because of the heat-sensitivity of their reproduction process. A full understanding of heat-stress (HS) response and thermotolerance of tomato reproduction is still lacking. Recently, using external application of the plant hormone ethylene, it was demonstrated that ethylene plays a role in heat-tolerance of tomato pollen (the male reproductive cells). In order to expand our understanding on involvement of ethylene in tomato pollen thermotolerance, we analyzed the response of wild type and ethylene-related tomato mutant plants to HS, at physiological and molecular levels. We report that mild chronic HS conditions highly reduce the number of viable and germinating pollen grains as well as the production of seeded fruits in wild type tomato plants, while no significant reduction was detected/observed in pollen quality, number of seeded fruits and seeds per fruit in plants of the ethylene over-producer mutant epinastic. Our findings suggest that ethylene is involved in thermotolerance of tomato reproduction, pointing to an effect on pollen viability and germination potential, highlighting candidate genes involved in pollen response to HS (like SlHSP17, SlHSP101, SlMBF1) and suggesting directions for further studies.

Tomato Pollen Wall Development, and the Role of FIN4 in the Formation of the Pollen Intine

Tomato Pollen Wall Development, and the Role of FIN4 in the Formation of the Pollen Intine

Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato.

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP's activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

Ruderal Plants Providing Bees Diversity on Rural Properties

Many are the anthropogenic drivers of pollinator decline, but the loss of suitable habitats, among other effects caused by agricultural intensification, deserves special attention. Reduction in the availability of floral resources negatively affects bee communities, compromising bee species composition, foraging behavior, corporal size, and fitness. Our study aims to understand whether the presence of herbaceous plants, acting as foraging sites, next to crops contributes to bee species richness in smallholder rural properties. Bee sampling was performed on smallholder rural properties in the municipality of Guapiara, southern São Paulo state. Individuals who visited the flowers of ruderal plants and crops were collected, using an entomological net, for ten months. A total of 61 bee species were identified, with the highest species richness being sampled in ruderal plant flowers in the three properties studied. Only in one property, ruderal plants hosted a more diverse bee assemblage (Shannon-Weiner and taxonomic diversity indices), but species composition differed from that sampled in crop plants (Jaccard index) in all properties. Thirty-two species were sampled exclusively in ruderal plants, versus 9 only in crops and 20 species in both types of plants. Pollen analysis showed that of the 22 species of bees that were sampled only on flowers of ruderal plants, 9 species carried pollen of tomato and one species of bee carried pollen of kabocha squash. Ruderal plants can provide an alternative food resource for pollinators, enabling these insects to remain in or be attracted to crop areas, where, in addition to visiting such plants, they also visit the cultivated plant flowers. Allowing coexistence between crops and ruderal plants, provided that the issues of plant health are observed, is a simple and low-cost measure for farmers and will provide both economic and environmental benefits.

Pollen Load Spectrum of Tomato Pollinators.

Vibrating bees are the main pollinators of the tomato plant (Solanum lycopersicum L.). Knowledge of other alternative food resources for these bees is fundamental for pollinator management actions in agricultural areas. The objective of this study was to evaluate the plants used as food resources for the main pollinators Bombus morio (Swederus) and Exomalopsis analis Spinola in plantation areas. The study was conducted in 12 plantation areas in São José de Ubá, southeastern Brazil, during the flowering period of S. lycopersicum. The pollen material contained on the hind legs of 40 B. morio females and 72 E. analis females was analyzed and compared with the reference slides made from 155 flowering plant species (35 botanical families) sampled close to the plantations. The pollen material was submitted to acetolysis and mounted in glycerin gelatin and analyzed under optical microscope. From B.morio corbiculae were identified 188 pollen types (52 identified from reference slides) and 189 types from E. analis scopae (54 in reference slides). Besides tomato pollen, other most abundant types belong to Fabaceae (8%) in B. morio samples, and Hyptis and Solanum sp in E. analis samples. The trophic niche overlap was close to zero when the tomato pollen was disregarded, indicating that both pollinators use distinct sources. The results confirm the generalist character of tomato pollinators; in addition, the use of floral resources from several other plants, even at tomato flowering peak, emphasizes the importance of maintaining flowering plant composition around agricultural areas.

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing.

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non-coding regions in the gene promoter and 5'UTR regions of genes. Poly-A/T and AT rich motifs were found enriched in 1kb promoter regions flanking the CO sites. Non-crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non-homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.

Optimizing pollencounter for high throughput phenotyping of pollen quality in tomatoes

The macro “PollenCounter” in ImageJ was initially developed to assess pollen viability in grapevine. We set out to see if PollenCounter could be used to assess pollen number and viability in tomatoes.•We tested different optimization scenarios by adjusting the pollen size (100–900, 200–900 pixel2) and circularity of pollen grains (0.4–1, 0.5–1, and 0.6–1) on 31 microscopic images of stained tomato pollen. Both total pollen number and proportion of viable pollen were positively and significantly correlated with the outputs from manual counting. The scenario with 100–900 pixel2 pollen size and 0.4–1 circularity had the highest association for pollen number (r = 0.99) and pollen viability (r = 0.86). PollenCounter is 32-fold faster than manual counting.•We added a command to the macro to automatically save the outputs containing the number of total and viable pollen, avoiding transcription errors inherent to manual counting.•We successfully applied the optimized PollenCounter to discriminate tomato genotypes based on pollen number and pollen viability under heat stress. Our results show that PollenCounter, as an open-access macro, can be customized and improved to meet users’ needs. The use of PollenCounter can save time and money in pollen quality assessment. We outline the steps to optimize the macro for other samples or crop species. The optimized macro could allow efficient screening of a large germplasm collection for pollen thermo-tolerance and selection of best thermo-tolerant individuals in breeding programs.

Tomato hybrid seed production using stored pollen grains: temperature and storage time interactions and their effects on pollen viability

In the past decades, tomato (Solanum lycopersicum L.) hybrid seed production has been massively employed in breeding programs throughout the world. However, some important physiological factors should be considered in order to attain adequate yield in association with high physiological and genetic qualities, during hybrid seed production. Pollen viability under stored conditions is of crucial importance or hybrid seed production as well as co-hybrid development programs and collaborative shuttle breeding networks among partner research institutions/companies. The main objective of the present work was to identify the best combinations of temperature and storage time of tomato pollen grains aiming to produce hybrid seeds with high genetic and physiological qualities. Pollen grain viability of the male tomato inbred line of the hybrid 'San Vito' (San Marzano type) was evaluated after the following storage conditions: 4, -20, and -80°C in combination with 30, 60, and 120 days of pollen storage. The viability of the pollen grains was evaluated by visualizing them in vivo through stigma tissue and counting the percentage of grains with conspicuous pollen tube growth (germination). The results indicated that the best storage condition for the tomato pollen was -20°C for 60 days. Under this storage condition, the average pollen germination was around 41%. These results reinforce that storage at low temperatures might represent an alternative technique for tomato pollen conservation, assisting the hybrid seed production systems and minimizing the risk associated with the potential lack of flowering synchronization between male and female parental inbred lines.

Downregulation of the auxin transporter gene SlPIN8 results in pollen abortion in tomato

SlPIN8 is expressed specifically within tomato pollen, and that it is involved in tomato pollen development and intracellular auxin homeostasis. The auxin (IAA) transport protein PIN-FORMED (PIN) plays key roles in various aspects of plant development. The biological role of the auxin transporter SlPIN8 in tomato development remains unclear. Here, we examined the expression pattern of the SlPIN8 gene in vegetative and reproductive organs of tomato. RNA interference (RNAi) transgenic lines specifically silenced for the SlPIN8 gene were generated to identify the role of SlPIN8 in pollen development. We found that SlPIN8 mRNA is expressed specifically within tomato pollen. In the anthers, the highest mRNA expression and β-glucuronidase (GUS) activity of promoter-SlPIN8-GUS was detected during late stages of anther development, when pollen maturation occurred. The downregulation of SlPIN8 did not drastically affect the vegetative growth of tomato. However, in SlPIN8-RNAi transgenic plants, approximately 80% of the pollen grains were identified to be abnormal and lack viability; they were shriveled and flattened. Furthermore, the downregulation of SlPIN8 affected the gene expression of some anther development-specific proteins. SlPIN8-RNAi transgenic plants induced seedless fruits because of defective pollen function rather than defective female gametophyte function. In addition, SlPIN8 was found to localize to the endoplasmic reticulum, consistent with the changes in the auxin levels of SlPIN8-RNAi lines, whereas the level of free IAA was increased in SlPIN8-overexpressing protoplasts, indicating that SlPIN8 is involved in intracellular auxin homeostasis.

The effect of isolation methods of tomato pollen on the results of metabolic profiling

IntroductionUntargeted metabolomics is a powerful tool to detect hundreds of metabolites within a given tissue and to compare the metabolite composition of samples in a comprehensive manner. However, with regard to pollen research such comprehensive metabolomics approaches are yet not well developed. To enable isolation of pollen that is tightly enclosed within the anthers of the flower, such as immature pollen, the current pollen isolation protocols require the use of a watery solution. These protocols raise a number of concerns for their suitability in metabolomics analyses, in view of possible metabolic activities in the pollen and contamination with anther metabolites.ObjectivesWe assessed the effect of different sample preparation procedures currently used for pollen isolation for their suitability to perform metabolomics of tomato pollen.MethodsPollen were isolated using different methods and the metabolic profiles were analysed by liquid chromatography–mass spectrometry (LC–MS).ResultsOur results demonstrated that pollen isolation in a watery solution led to (i) rehydration of the pollen grains, inducing marked metabolic changes in flavonoids, phenylpropanoids and amino acids and thus resulting in a metabolite profile that did not reflect the one of mature dry pollen, (ii) hydrolysis of sucrose into glucose and fructose during subsequent metabolite extraction, unless the isolated and rehydrated pollen were lyophilized prior to extraction, and (iii) contamination with anther-specific metabolites, such as alkaloids, thus compromising the metabolic purity of the pollen fraction.ConclusionWe conclude that the current practices used to isolate pollen are suboptimal for metabolomics analyses and provide recommendations on how to improve the pollen isolation protocol, in order to obtain the most reliable metabolic profile from pollen tissue.

Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains.

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.