Transcriptional profiling of maturing tomato (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the heat stress response

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

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

Water status and associated processes marks critical stages in pollen development and functioning

3The 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 ntomophilous 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.reservedmixedFiron N.; Nepi M.; Pacini E.Firon, N.; Nepi, Massimo; Pacini, Ettor

Ethylene accumulation in 'Beauregard' sweetpotato cuttings.

<p>Ethylene accumulation by ‘Beauregard’ cuttings subjected to 1-methylcyclopropene (1-MCP) treatment in sealed containers. From Villordon, A., Clark, C., LaBonte, D., & Firon, N. (2012). 1-Methylcyclopropene has a variable effect on adventitious root emergence from cuttings of two sweetpotato cultivars. HortScience, 47(12), 1764-1767.</p

Survey of Genes Involved in Biosynthesis, Transport, and Signaling of Phytohormones with Focus on

Phytohormones control the development and growth of plants, as well as their response to biotic and abiotic stress. The seven most well-studied phytohormone classes defined today are as follows: auxins, ethylene, cytokinin, abscisic acid, jasmonic acid, gibberellins, and brassinosteroids. The basic principle of hormone regulation is conserved in all plants, but recent results suggest adaptations of synthesis, transport, or signaling pathways to the architecture and growth environment of different plant species. Thus, we aimed to define the extent to which information from the model plant Arabidopsis thaliana is transferable to other plants such as Solanum lycopersicum. We extracted the co-orthologues of genes coding for major pathway enzymes in A. thaliana from the translated genomes of 12 species from the clade Viridiplantae. Based on predicted domain architecture and localization of the identified proteins from all 13 species, we inspected the conservation of phytohormone pathways. The comparison was complemented by expression analysis of (co-) orthologous genes in S. lycopersicum. Altogether, this information allowed the assignment of putative functional equivalents between A. thaliana and S. lycopersicum but also pointed to some variations between the pathways in eudicots, monocots, mosses, and green algae. These results provide first insights into the conservation of the various phytohormone pathways between the model system A. thaliana and crop plants such as tomato. We conclude that orthologue prediction in combination with analysis of functional domain architecture and intracellular localization and expression studies are sufficient tools to transfer information from model plants to other plant species. Our results support the notion that hormone synthesis, transport, and response for most part of the pathways are conserved, and species-specific variations can be found

Manipulating Root System Architecture in Sweetpotato for Global Food Security: Progress, Prospects, and Applications

<p>Biotic and abiotic variables that influence sweetpotato root architecture development.</p

Proximal and Distal Parts of Sweetpotato Adventitious Roots Display Differences in Root Architecture, Lignin, and Starch Metabolism and Their Developmental Fates

Sweetpotato is an important food crop globally, serving as a rich source of carbohydrates, vitamins, fiber, and micronutrients. Sweetpotato yield depends on the modification of adventitious roots into storage roots. The underlying mechanism of this developmental switch is not fully understood. Interestingly, storage-root formation is manifested by formation of starch-accumulating parenchyma cells and bulking of the distal part of the root, while the proximal part does not show bulking. This system, where two parts of the same adventitious root display different developmental fates, was used by us in order to better characterize the anatomical, physiological, and molecular mechanisms involved in sweetpotato storage-root formation. We show that, as early as 1 and 2 weeks after planting, the proximal part of the root exhibited enhanced xylem development together with increased/massive lignin deposition, while, at the same time, the distal root part exhibited significantly elevated starch accumulation. In accordance with these developmental differences, the proximal root part exhibited up-regulated transcript levels of sweetpotato orthologs of Arabidopsis vascular-development regulators and key genes of lignin biosynthesis, while the distal part showed up-regulation of genes encoding enzymes of starch biosynthesis. All these recorded differences between proximal and distal root parts were further enhanced at 5 weeks after planting, when storage roots were formed at the distal part. Our results point to down-regulation of fiber formation and lignification, together with up-regulation of starch biosynthesis, as the main events underlying storage-root formation, marking/highlighting several genes as potential regulators, providing a valuable database of genes for further research

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

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.</p