Gene expression regulation during tissue development is extremely complex. A key mechanism of gene regulation is the recognition of regulatory motifs, also known as cis-regulatory elements (CREs), by various proteins in gene promoter regions. Localization of these motifs near the transcription start site (TSS) or translation start site (ATG) is crucial for transcription initiation and rate. Transcription levels of individual genes, regulated by these motifs, can vary significantly across tissues and developmental stages, especially in processes like sexual reproduction. However, the precise localization and visualization of these motifs in relation to gene expression in specific tissues can be challenging. Here, we introduce a freely available tool called GOLEM (Gene regulatOry eLEMents; https://golem.ncbr.muni.cz), which enables users to precisely locate any motif of interest with respect to TSS or ATG within the relevant plant genomes across the plant Tree of Life (Chara, Marchantia, Physcomitrium, Azolla, Ceratopteris, Amborella, Oryza, Zea, Solanum and Arabidopsis). The visualization of the motifs is performed with respect to the transcript levels of particular genes in leaves and male reproductive tissues and can be compared with genome-wide distribution regardless of the transcription level. Additionally, genes with specific CREs at defined positions and high expression in selected tissues can be exported for further analysis. GOLEM's functionality is illustrated by its application to conserved motifs (e.g. TATA-box, ABRE, I-box, and TC-element), hormone-responsive elements (GCC-box, ARR10_binding motif), as well as to male gametophyte-related motifs (e.g., LAT52, MEF2, and DOF_core).

• Keywords
• GOLEM, Gene regulatOry eLEMents, TSS, gametophyte, motif localization, plant genes, promoter elements, technical advance,
• MeSH
• Arabidopsis genetics   MeSH
• Genome, Plant genetics   MeSH
• Transcription Initiation Site MeSH
• Promoter Regions, Genetic * genetics   MeSH
• Pollen * genetics   MeSH
• Gene Expression Regulation, Plant genetics   MeSH
• Software * MeSH
• Publication type
• Journal Article MeSH

Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

• Keywords
• Arabidopsis, Male gametophyte, Microgametogenesis, Proteome, RNA-seq,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Proteomics MeSH
• Pollen genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Reproducibility of Results MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arabidopsis Proteins * MeSH

Plant microgametogenesis involves stages leading to the progressive development of unicellular microspores into mature pollen. Despite the active and continuing interest in the study of male reproductive development, little is still known about the hormonomics at each ontogenetic stage. In this work, we characterized the profiles and dynamics of phytohormones during the process of microgametogenesis in four Nicotiana species (Nicotiana tabacum, Nicotiana alata, Nicotiana langsdorffii, and Nicotiana mutabilis). Taking advantage of advanced HPLC-ESI-MS/MS, twenty to thirty endogenous hormone derivatives were identified throughout pollen ontogenesis, including cytokinins, auxins, ABA and its derivatives, jasmonates, and phenolic compounds. The spectra of endogenous phytohormones changed dynamically during tobacco pollen ontogeny, indicating their important role in pollen growth and development. The different dynamics in the accumulation of endogenous phytohormones during pollen ontogenesis between N. tabacum (section Nicotiana) and the other three species (section Alatae) reflects their different phylogenetic positions and origin within the genus Nicotiana. We demonstrated the involvement of certain phytohormone forms, such as cis-zeatin- and methylthiol-type CKs, some derivatives of abscisic acid, phenylacetic and benzoic acids, in pollen development for the first time here. Our results suggest that unequal levels of endogenous hormones and the presence of specific derivatives may be characteristic for pollen development in different phylogenetic plant groups. These results represent the currently most comprehensive study of plant hormones during the process of pollen development.

• Keywords
• Nicotiana spp., hormonome, male gametophyte, ontogeny, phytohormones, pollen development,
• Publication type
• Journal Article MeSH

Being rooted in place, plants are faced with the challenge of responding to unfavourable local conditions. One such condition, heat stress, contributes massively to crop losses globally. Heatwaves are predicted to increase, and it is of vital importance to generate crops that are tolerant to not only heat stress but also to several other abiotic stresses (e.g. drought stress, salinity stress) to ensure that global food security is protected. A better understanding of the molecular mechanisms that underlie the temperature stress response in pollen will be a significant step towards developing effective breeding strategies for high and stable production in crop plants. While most studies have focused on the vegetative phase of plant growth to understand heat stress tolerance, it is the reproductive phase that requires more attention as it is more sensitive to elevated temperatures. Every phase of reproductive development is affected by environmental challenges, including pollen and ovule development, pollen tube growth, male-female cross-talk, fertilization, and embryo development. In this review we summarize how pollen is affected by heat stress and the molecular mechanisms employed during the stress period, as revealed by classical and -omics experiments.

• Keywords
• heat stress (HS), heat stress response (HSR), multiomics, pollen development, thermotolerance,
• MeSH
• Stress, Physiological MeSH
• Pollen MeSH
• Heat-Shock Response MeSH
• Plant Breeding * MeSH
• Thermotolerance * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Analyses of secretomes of in vitro grown pollen tubes from Amborella, maize and tobacco identified many components of processes associated with the cell wall, signaling and metabolism as well as novel small secreted peptides. Flowering plants (angiosperms) generate pollen grains that germinate on the stigma and produce tubes to transport their sperm cells cargo deep into the maternal reproductive tissues toward the ovules for a double fertilization process. During their journey, pollen tubes secrete many proteins (secreted proteome or secretome) required, for example, for communication with the maternal reproductive tissues, to build a solid own cell wall that withstands their high turgor pressure while softening simultaneously maternal cell wall tissue. The composition and species specificity or family specificity of the pollen tube secretome is poorly understood. Here, we provide a suitable method to obtain the pollen tube secretome from in vitro grown pollen tubes of the basal angiosperm Amborella trichopoda (Amborella) and the Poaceae model maize. The previously published secretome of tobacco pollen tubes was used as an example of eudicotyledonous plants in this comparative study. The secretome of the three species is each strongly different compared to the respective protein composition of pollen grains and tubes. In Amborella and maize, about 40% proteins are secreted by the conventional "classic" pathway and 30% by unconventional pathways. The latter pathway is expanded in tobacco. Proteins enriched in the secretome are especially involved in functions associated with the cell wall, cell surface, energy and lipid metabolism, proteolysis and redox processes. Expansins, pectin methylesterase inhibitors and RALFs are enriched in maize, while tobacco secretes many proteins involved, for example, in proteolysis and signaling. While the majority of proteins detected in the secretome occur also in pollen grains and pollen tubes, and correlate in the number of mapped peptides with relative gene expression levels, some novel secreted small proteins were identified. Moreover, the identification of secreted proteins containing pro-peptides indicates that these are processed in the apoplast. In conclusion, we provide a proteome resource from three distinct angiosperm clades that can be utilized among others to study the localization, abundance and processing of known secreted proteins and help to identify novel pollen tube secreted proteins for functional studies.

• Keywords
• Amborella, CRP, Cell wall, Maize, Pollen tube, Proteomics, Secretome, Signaling, Tobacco,
• MeSH
• Zea mays MeSH
• Magnoliopsida * MeSH
• Peptides MeSH
• Pollen Tube * MeSH
• Nicotiana MeSH
• Ovule MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Peptides MeSH

Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.

• MeSH
• Arabidopsis genetics   growth & development   MeSH
• Biological Evolution MeSH
• Germination genetics   physiology   MeSH
• Magnoliopsida genetics   growth & development   MeSH
• Proteomics MeSH
• Pollen genetics   growth & development   MeSH
• Pollen Tube genetics   growth & development   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Plant MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

• Keywords
• AFCVd propagation and eradication, Nicotiana tabacum, Proteome, RNA sequencing, RT qPCR, male gametophyte, viroid degradation, viroid replication,
• MeSH
• Cell Differentiation * MeSH
• Plant Diseases virology   MeSH
• Proteomics * MeSH
• Pollen * metabolism   virology   MeSH
• Plant Viruses metabolism   MeSH
• Gene Expression Profiling * MeSH
• Nicotiana * metabolism   virology   MeSH
• Viroids metabolism   MeSH
• Publication type
• Journal Article MeSH

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.

• MeSH
• Polyribosomes genetics   metabolism   MeSH
• Proteome genetics   metabolism   MeSH
• Proteomics methods   MeSH
• Pollen genetics   growth & development   metabolism   MeSH
• Pollen Tube genetics   growth & development   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Ribonucleoproteins genetics   metabolism   MeSH
• Ribosomes genetics   metabolism   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Gene Expression Profiling methods   MeSH
• Nicotiana genetics   growth & development   metabolism   MeSH
• Gene Expression Regulation, Developmental MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH
• Ribonucleoproteins MeSH
• Plant Proteins MeSH

Callose is a plant-specific polysaccharide (β-1,3-glucan) playing an important role in angiosperms in many developmental processes and responses to biotic and abiotic stresses. Callose is synthesised at the plasma membrane of plant cells by callose synthase (CalS) and, among others, represents the main polysaccharide in the callose wall surrounding the tetrads of developing microspores and in the growing pollen tube wall. CalS proteins involvement in spore development is a plesiomorphic feature of terrestrial plants, but very little is known about their evolutionary origin and relationships amongst the members of this protein family. We performed thorough comparative analyses of callose synthase family proteins from major plant lineages to determine their evolutionary history across the plant kingdom. A total of 1211 candidate CalS sequences were identified and compared amongst diverse taxonomic groups of plants, from bryophytes to angiosperms. Phylogenetic analyses identified six main clades of CalS proteins and suggested duplications during the evolution of specialised functions. Twelve family members had previously been identified in Arabidopsis thaliana. We focused on five CalS subfamilies directly linked to pollen function and found that proteins expressed in pollen evolved twice. CalS9/10 and CalS11/12 formed well-defined clades, whereas pollen-specific CalS5 was found within subfamilies that mostly did not express in mature pollen vegetative cell, although were found in sperm cells. Expression of five out of seven mature pollen-expressed CalS genes was affected by mutations in bzip transcription factors. Only three subfamilies, CalS5, CalS10, and CalS11, however, formed monophyletic, mostly conserved clades. The pairs CalS9/CalS10, CalS11/CalS12 and CalS3 may have diverged after angiosperms diversified from lycophytes and bryophytes. Our analysis of fully sequenced plant proteins identified new evolutionary lineages of callose synthase subfamilies and has established a basis for understanding their functional evolution in terrestrial plants.

• MeSH
• Phylogeny MeSH
• Glucosyltransferases genetics   MeSH
• Evolution, Molecular * MeSH
• Arabidopsis Proteins genetics   MeSH
• Pollen * MeSH
• Genes, Plant MeSH
• Transcription Factors genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1,3-beta-glucan synthase MeSH Browser
• Glucosyltransferases MeSH
• Arabidopsis Proteins MeSH
• Transcription Factors MeSH

In tobacco, three sequence variants of the TERT gene have been described. We revealed unbalanced levels of TERT variant transcripts in vegetative tobacco tissues and enhanced TERT transcription and telomerase activity in reproductive tissues. Telomerase is a ribonucleoprotein complex responsible for the maintenance of telomeres, structures delimiting ends of linear eukaryotic chromosomes. In the Nicotiana tabacum (tobacco) allotetraploid plant, three sequence variants (paralogs) of the gene coding for the telomerase reverse transcriptase subunit (TERT) have been described, two of them derived from the maternal N. sylvestris genome (TERT_Cs, TERT_D) and one originated from the N. tomentosiformis paternal genome (TERT_Ct). In this work, we analyzed the transcription of TERT variants in correlation with telomerase activity in tobacco tissues. High and approximately comparable levels of TERT_Ct and TERT_Cs transcripts were detected in seedlings, roots, flower buds and leaves, while the transcript of the TERT_D variant was markedly underrepresented. Similarly, in N. sylvestris tissues, TERT_Cs transcript significantly predominated. A specific pattern of TERT transcripts was found in samples of tobacco pollen with the TERT_Cs variant clearly dominating particularly at the early stage of pollen development. Detailed analysis of TERT_C variants representation in functionally distinct fractions of pollen transcriptome revealed their prevalence in large ribonucleoprotein particles encompassing translationally silent mRNA; only a minority of TERT_Ct and TERT_Cs transcripts were localized in actively translated polysomes. Histones of the TERT_C chromatin were decorated predominantly with the euchromatin-specific epigenetic modification in both telomerase-positive and telomerase-negative tobacco tissues. We conclude that the existence and transcription pattern of tobacco TERT paralogs represents an interesting phenomenon and our results indicate its functional significance. Nicotiana species have again proved to be appropriate and useful model plants in telomere biology studies.

• Keywords
• Gene sequence variant, Pollen, Polyploids, Telomerase, Telomere, Transcription,
• MeSH
• Cell Nucleus genetics   MeSH
• Chromatin Immunoprecipitation MeSH
• Euchromatin metabolism   MeSH
• Transcription, Genetic MeSH
• Genetic Variation * MeSH
• Histones metabolism   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Organ Specificity genetics   MeSH
• Polyribosomes metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Pollen Tube growth & development   MeSH
• Gene Expression Regulation, Plant * MeSH
• Nicotiana genetics   MeSH
• Telomerase genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Euchromatin MeSH
• Histones MeSH
• RNA, Messenger MeSH
• Telomerase MeSH