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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 grains have the capacity for ethylene production, possessing specific components of the ethylene-biosynthesis and -signaling pathways, being affected/responsive to high-temperature conditions. Exposure of plants to heat stress (HS) conditions reduces crop yield and quality, mainly due to sensitivity of pollen grains. Recently, it was demonstrated that ethylene, a gaseous plant hormone, plays a significant role in tomato pollen heat-tolerance. It is not clear, however, whether, or to what extent, pollen grains are dependent on the capacity of the surrounding anther tissues for ethylene synthesis and signaling, or can synthesize this hormone and possess an active signaling pathway. The aim of this work was (1) to investigate if isolated, maturing and mature, tomato pollen grains have the capacity for ethylene production, (2) to find out whether pollen grains possess an active ethylene-biosynthesis and -signaling pathway and characterize the respective tomato pollen components at the transcript level, (3) to look into the effect of short-term HS conditions. Results from accumulation studies showed that pollen, anthers, and flowers produced ethylene and HS affected differentially ethylene production by (rehydrated) mature pollen, compared to anthers and flowers, causing elevated ethylene levels. Furthermore, several ethylene synthesis genes were expressed, with SlACS3 and SlACS11 standing out as highly HS-induced genes of the pollen ethylene biosynthesis pathway. Specific components of the ethylene-signaling pathway as well as several ethylene-responsive factors were expressed in pollen, with SlETR3 (ethylene receptor; named also NR, for never ripe) and SlCTR2 (constitutive triple response2) being HS responsive. This work shows that tomato pollen grains have the capacity for ethylene production, possessing active ethylene-biosynthesis and -signaling pathways, highlighting specific pollen components that serve as a valuable resource for future research on the role of ethylene in pollen thermotolerance.

Pollen development at high temperature depends on a fine balance between acclimation and injury.

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.

Introduction Untargeted 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. Objectives We assessed the effect of different sample preparation procedures currently used for pollen isolation for their suitability to perform metabolomics of tomato pollen. Methods Pollen were isolated using different methods and the metabolic profiles were analysed by liquid chromatography–mass spectrometry (LC–MS). Results Our 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. Conclusion We 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. Electronic supplementary material The online version of this article (10.1007/s11306-018-1471-4) contains supplementary material, which is available to authorized users.

The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.

Pollen thermotolerance. Global warming is predicted to increase the frequency and severity of extreme weather phenomena such as heat waves thereby posing a major threat for crop productivity and food security. The yield in case of most crop species is dependent on the success of reproductive development. Pollen development has been shown to be highly sensitive to elevated temperatures while the development of the female gametophyte as well as sporophytic tissues might also be disturbed under mild or severe heat stress conditions. Therefore, assessing pollen thermotolerance is currently of high interest for geneticists, plant biologists and breeders. A key aspect in pollen thermotolerance studies is the selection of the appropriate heat stress regime, the developmental stage that the stress is applied to, as well as the method of application. Literature search reveals a rather high variability in heat stress treatments mainly due to the lack of standardized protocols for different plant species. In this review, we summarize and discuss experimental approaches that have been used in various crops, with special focus on tomato, rice and wheat, as the best studied crops regarding pollen thermotolerance. The overview of stress treatments and the major outcomes of each study aim to provide guidelines for similar research in other crops.

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.

Exposure to high temperatures (heat stress) causes reduced yield in tomatoes (Lycopersicon esculentum), mainly by affecting male gametophyte development. Two experiments were conducted where several tomato cultivars were grown under heat stress, in growth chambers (day/night temperatures of 31/25 °C) or in greenhouses (day/night temperatures of 32/26 °C), or under control (day/night temperatures of 28/22 °C) conditions. In heat-sensitive cultivars, heat stress caused a reduction in the number of pollen grains, impaired their viability and germinability, caused reduced fruit set and markedly reduced the numbers of seeds per fruit. In the heat-tolerant cultivars, however, the number and quality of pollen grains, the number of fruits and the number of seeds per fruit were less affected by high temperatures. In all the heat-sensitive cultivars, the heat-stress conditions caused a marked reduction in starch concentration in the developing pollen grains at 3 days before anthesis, and a parallel decrease in the total soluble sugar concentration in the mature pollen, whereas in the four heat-tolerant cultivars tested, starch accumulation at 3 days before anthesis and soluble sugar concentration at anthesis were not affected by heat stress. These results indicate that the carbohydrate content of developing and mature tomato pollen grains may be an important factor in determining pollen quality, and suggest that heat-tolerant cultivars have a mechanism for maintaining the appropriate carbohydrate content under heat stress.

SummaryExposure to high temperatures causes reduced yields in tomato (Lycopersicon esculentum), mainly by affecting male gametophyte development. The effect of heat stress on pollen characteristics...

The paper supports the view that ethylene plays a significant role in maintaining tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels and increased tomato pollen sensitivity to heat stress. On the other hand, increasing ethylene levels before heat-stress improved pollen quality., Background and aims Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene involvement in the pollen heat-stress response. Here we elucidated ethylene's involvement in pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling pathway and altering ethylene levels on tomato pollen functioning under heat stress. Methodology Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene response sensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to control or heat-stress growing conditions, and pollen quality was determined. Starch and carbohydrates were measured in isolated pollen grains from these plants. The effect of pretreating cv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser or inhibitor of ethylene biosynthesis on pollen quality was assessed. Principal results Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant reduction in the total number of pollen grains, reduction in the number of viable pollen and elevation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollen grains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatment of tomato plants with an ethylene releaser increased pollen quality under heat stress, with an over 5-fold increase in the number of germinating pollen grains per flower. Pretreatment with an ethylene biosynthesis inhibitor reduced the number of germinating pollen grains following heat-stress exposure over 5-fold compared with non-treated controls. Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylene signalling pathway or reducing ethylene levels increased tomato pollen sensitivity to heat stress, whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.

The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant--the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration--the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion--the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods--the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination--the pollen-stigma interaction phase.This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability.Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions.

Calcium phosphate-adsorbed allergen extracts are used for subcutaneous immunotherapy to avoid the use of aluminium adjuvants.A double-blind, placebo-controlled study was performed in order to confirm the safety and assess the efficacy of a standardized five-grass-pollen extract adsorbed onto calcium phosphate for specific immunotherapy (IT).Twenty-nine patients with seasonal rhinoconjunctivitis were randomized to receive either the active preparation (16 patients) or placebo (13 patients), in a 1-year study. During the increasing dose phase, an extract ranging from 0.1 IR per ml to 50 IR per ml was administered at a rate of one subcutaneous injection per week until a maintenance dose was reached. The patients were assessed by symptom diary and rescue medications during seasonal exposure and specific nasal and skin reactivity before and after IT. Immunological parameters (specific IgE and IgG4 antibodies) were assessed before, during and after IT.The overall symptoms score (mean AUC) was not significantly different between the IT group and the placebo group during grass-pollen exposure (49.6 vs. 56, respectively). The total medication score (mean AUC) was significantly lower in the IT group than in the placebo group (11 vs. 41, P0.01, Mann-Whitney U-test). The cumulative symptom/medication score was significantly lower in the IT group than in the placebo group (64.5 vs. 102.3, P0.05, U-test). A significant increase in nasal reactivity threshold was observed after IT in the IT group (21. 4 IR/mL before IT vs. 63.4 IR/mL after IT, P0.01, Wilcoxon), whereas no significant changes were observed in the placebo group (31.0 IR/mL before IT vs. 37.7 IR/mL after IT). IT induced a significant reduction in grass pollen cutaneous reactivity in the actively treated group (P0.001). A significant increase in serum-specific IgG4 antibody response was observed in the IT group (3.1% before IT vs. 10.1% after IT, P0.001). Nine patients in the IT group developed moderate immediate systemic reactions vs. two patients in the placebo group.Specific immunotherapy with calcium phosphate-adsorbed standardized grass pollen extract was safe and effective for the treatment of patients with seasonal allergic rhinoconjunctivitis.

Exposing pepper (Capsicum annuum) plants to extremely high day temperatures (HDT) (day/night temperatures of 36 ± 2/10 ± 2°C), obtained by keeping the greenhouse closed during the day to exploit solar heating, prevented the development of low night temperature (LNT) symptoms. Plants of cultivars Fiesta and Selica grown under LNTs (10 ± 2°C) and moderate day temperatures (25 ± 2°C) during winter exhibited retarded growth, reduced leaf numbers, and deformed fruits with few or no seeds. LNT caused a reduction in the number and quality of pollen grains: the reduction in pollen quality was associated with reduced starch accumulation in pollen grains at 3 days before anthesis (DBA) and a decrease of more than two-fold in total soluble sugars in the mature pollen grains. This inhibitory effect was associated with more than 50% reduction in the enzymatic activities of the cell wall-bound and soluble acid invertases that catalyze the hydrolysis of incoming sucrose molecules. All these symptoms were prevented by HDT treatment which matched the vegetative and reproductive performance of the plants to those of plants grown under optimal night temperature (ONT) conditions (day/night temperatures of 23 ± 2/18 ± 2°C). HDT also prevented the inhibitory effect of LNT on enzymatic activities of both invertases in pollen at 5 DBA and brought about the accumulation of high levels of starch in pollen at 3 DBA. The results presented could support the development of a novel procedure for producing greenhouse crops with minimum or even with no fuel consumption for heating during the winter nights in regions with bright and sunny days.