Věra Čapková

Comprehensive analysis of tobacco pollen transcriptome unveils common pathways in polar cell expansion and underlying heterochronic shift during spermatogenesis

BackgroundMany flowering plants produce bicellular pollen. The two cells of the pollen grain are destined for separate fates in the male gametophyte, which provides a unique opportunity to study genetic interactions that govern guided single-cell polar expansion of the growing pollen tube and the coordinated control of germ cell division and sperm cell fate specification. We applied the Agilent 44 K tobacco gene chip to conduct the first transcriptomic analysis of the tobacco male gametophyte. In addition, we performed a comparative study of the Arabidopsis root-hair trichoblast transcriptome to evaluate genetic factors and common pathways involved in polarized cell-tip expansion.ResultsProgression of pollen grains from freshly dehisced anthers to pollen tubes 4 h after germination is accompanied with > 5,161 (14.9%) gametophyte-specific expressed probes active in at least one of the developmental stages. In contrast, > 18,821 (54.4%) probes were preferentially expressed in the sporophyte. Our comparative approach identified a subset of 104 pollen tube-expressed genes that overlap with root-hair trichoblasts. Reverse genetic analysis of selected candidates demonstrated that Cu/Zn superoxide dismutase 1 (CSD1), a WD-40 containing protein (BP130384), and Replication factor C1 (NtRFC1) are among the central regulators of pollen-tube tip growth. Extension of our analysis beyond the second haploid mitosis enabled identification of an opposing-dynamic accumulation of core regulators of cell proliferation and cell fate determinants in accordance with the progression of the germ cell cycle.ConclusionsThe current study provides a foundation to isolate conserved regulators of cell tip expansion and those that are unique for pollen tube growth to the female gametophyte. A transcriptomic data set is presented as a benchmark for future functional studies using developing pollen as a model. Our results demonstrated previously unknown functions of certain genes in pollen-tube tip growth. In addition, we highlighted the molecular dynamics of core cell-cycle regulators in the male gametophyte and postulated the first genetic model to account for the differential timing of spermatogenesis among angiosperms and its coordination with female gametogenesis.

Expression of dehydrin 5 during the development of frost tolerance in barley (Hordeum vulgare).

The Dhn5 gene is the major cold-inducible dehydrin gene in barley. This study deals with the relationship between Dhn5 gene expression and its protein product accumulation, and the development of frost tolerance (FT) upon cold acclimation (CA) in 10 barley cultivars of different growth habits and geographical origins. The activation of Dhn5 gene expression was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the accumulation of DHN5 protein was evaluated by protein gel blot analysis using a specific anti-dehydrin antibody, and the acquired level of FT was determined by a direct frost test. During the first 2 weeks of CA, there was a rapid increase in Dhn5 gene expression, DHN5 protein accumulation and FT in all cultivars examined. After 2 weeks of CA, differences in DHN5 accumulation and in FT measured as lethal temperature (LT(50)) were observed between the cultivars belonging to different growth habits. Specifically, intermediate (I) and winter (W) cultivars showed a higher level of DHN5 accumulation and FT than the spring (S) cultivars, which exhibited a lower level of accumulated DHN5 and FT. (Intermediate cultivars do not have vernalization requirement, but they are able to induce a relatively high level of FT upon CA.) In contrast, no differences between the cultivars belonging to different growth habits in Dhn5 mRNA accumulation were found. After 3 weeks of CA, the differences in accumulated DHN5 and FT between the individual growth habits became evident due to different developmental regulation of FT. The amount of accumulated DHN5 corresponded well with the level of FT of individual cultivars. We conclude that the amount of accumulated DHN5 after a certain period of CA differed according to the growth habits of cultivars and can be used as a marker for determination of FT in barley.

Cytoskeleton-associated large RNP complexes in tobacco male gametophyte (EPPs) are associated with ribosomes and are involved in protein synthesis, processing, and localization.

The progamic phase of male gametophyte development involves activation of synthetic and catabolic processes required for the rapid growth of the pollen tube. It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs. Here, we report the first structural and proteomic data about large ribonucleoprotein particles (EPPs) in tobacco male gametophyte. These complexes are formed in immature pollen where they contain translationally silent mRNAs. Although massively activated at the early progamic phase, they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region. Moreover, EPPs were shown to contain ribosomal subunits, rRNAs and a set of mRNAs. Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage, transport and subsequent controlled activation resulting in protein synthesis, processing and precise localization. Such an organization is extremely useful in fast tip-growing pollen tube. There, massive and orchestrated protein synthesis, processing, and transport must take place in accurately localized regions. Moreover, presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too. Expression of vast majority of the closest orthologues of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis, the model species with advanced tricellular pollen.

Revealing phosphoproteins playing role in tobacco pollen activated in vitro

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co‐exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.

Impact of in vitro Cultivation Conditions on Stress Responses and on Changes in Thylakoid Membrane Proteins and Pigments of Tobacco during ex vitro Acclimation

Four physiologically and phenotypically diversified tobacco (Nicotiana tabacum L. cv. Samsun) plantlet variants had been generated by cultivation on media either lacking or containing sucrose (0 and 3 %, m/v) under two different photon flux densities (PFD), 50 µmol m−2 s−1 (LL) and 200 µmol m−2 s−1 (HL). Plantlets were transferred into soil without any pre-acclimation and grown either under PFD of 200 µmol m−2 s−1 or 700 µmol m−2 s−1. Sucrose feeding in vitro resulted in reduced degree and duration of wilting after transfer. The highest readiness for ex vitro acclimation was found in 3 % HL plants, in which changes of photosynthetic apparatus and stress responses were the smallest. On the contrary, the steepest decline of Fv/Fm ratio on the first day after transplantation, doubled chlorophyll content and almost tripled D1/LHC 2 ratio after 7 d of ex vitro growth under 700 µmol m−2 s−1 characterized 0 % HL plants, which had suffered chronic photoinhibition in vitro. Remarkably high abscisic acid content at the end of in vitro cultivation and during acclimation as well as increased synthesis of both D1 and LHC 2 proteins even at the end of analyzed acclimation period were found only in 0 % LL plants. Increase of D1/LHC 2 ratio and chlorophyll contents demonstrate that in vitro developed leaves of all plant variants are able to acclimate to new environment. The most surprising result in the whole study is the drop of D1 protein synthesis in all plants on the 3rd day. Five times decline of photoprotection level of xanthophylls in plants after ex vitro transfer into the same PFD showed stress character of in vitro cultures.

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 μmol m−2 s−1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 μmol m−2 s−1 during further ex vitro growth (in the period of 7th – 17th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 μmol m−2 s−1 (3 % LL) or without sucrose either under PFD of 50 μmol m−2 s−1 or 200 μmol m−2 s−1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.

Changes in Glycoprotein Patterns Associated with Male Gametophyte Development and with Induction of Pollen Embryogenesis in Nicotiana tabacum L.

Summary Patterns of glycoproteins (GPs) were examined during male gametophyte development in the anthers or in vitro and during induction of embryogenic P-grains by starvation treatment of young pollen. GPs extracted from soluble (post-mitochondrial) and insoluble fraction of microspores, pollen or P-grains were analyzed by SDS-PAGE and detected on blots by peroxidase-conjugated Concanavalin A (ConA). GP patterns of six stages from late microspores to pollen one day before shedding showed two sequences of gametophytic development in the anthers corresponding to pollen differentiation and pollen maturation. A number of preexisting GPs varied in staining intensity, a few bands disappeared and some GPs were newly synthesized. Changes in GP patterns occurred mainly in the endomembrane-containing soluble fraction. Microspores and young vacuolized pollen were particularly marked by a dominant specific 88 kDa GP, most qualitative changes were detectable at the end of differentiation and the phase of maturation was characterized by increasing staining intensity of most GPs, especially of bands 48, 59, 70 and 83 kDa and of new GPs 51, 53 and 75 kDa. Maturation also resulted in a marked increase of insoluble GPs, particularly of GPs 53, 59, 66 and in the range 70–83 kDa. The phases of differentiation and maturation correlated, respectively, with an increase and decrease of fresh mass of the anthers. Similar changes as in the anthers occurred during cultivation of isolated pollen in vitro with induction of maturation by lowering of the water potential of culture medium. Starvation treatment of differentiating pollen prevented further changes associated with gametophytic development and resulted in a strong decrease of GP levels. P-grains were characterized by absence of GPs larger than 78 kDa and presence of new relatively abundant GPs 58, 60 and 68 kDa. Unlike to ConA-binding GP patterns, no evident differences could be detected in the patterns of Coomassie blue stained proteins between young pollen, in vitro matured pollen and P-grains. Protein glycosylation might thus play specific roles in pollen differentiation and maturation and the starvation-induced GP changes could be associated with pollen dedifferentiation and the acquisition of the embryogenic competence.

Phosphoproteomics profiling of tobacco mature pollen and pollen activated in vitro

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

In search of ligands and receptors of the pollen tube: the missing link in pollen tube perception

The journey undertaken by the pollen tube in angiosperms to reach the deeply embedded female gametophyte for fertilization involves persistent guidance by the female gametophyte and accurate perception of the signals by the pollen tube. Several ovule-secreted peptides have been identified. Nevertheless, there are no exact findings on how these signals are perceived by the pollen tube. As a novel approach, we have improvised a modified SIV (semi-in vivo) technique, SIV-PS (SIV pollen tube secretome), to perform gel-free LC-MS/MS for high-throughput analysis of pollen-tube-secreted proteins. Our approach has led to the identification of over 1400 protein groups. Among them are pollen-tube-secreted ligands and receptor proteins representing potential male components in perceiving ovule-emitted cues for guidance. The present article reviews the missing link in pollen tube perception and showcases the improvised SIV-PS as a tool for high-throughput and targeted study of the pollen tube secretome.