Tube Prague Mature NEW!
Prague pneumatic post (Czech: Pražská potrubní pošta) is the world's last preserved municipal pneumatic post system. It is an underground system of metal tubes under the wider centre of Prague, totaling about 55 km (34 miles) in length. The system started service in 1889 and remained in use by the government, banks and the media until it was rendered inoperative by the August 2002 European floods.
tube prague mature
The system was established for those desiring to send a document quickly. The document would be taken to the post office and rolled up into a metal capsule. The clerk would then drop the metal capsule down a hatch leading to a predestined location. After the clerk pressed a button, the capsule would be moved by compressed air along a network of tubes beneath the pavement.
Therefore, it is no surprise that a number of pollen mRNAs were shown to accumulate in pollen-stored ribonucleoprotein (RNP) particles, which remain intact even in a buffer comprising strong detergents. This novel class of detergent-resistant RNP particles was annotated as EDTA/puromycin-resistant particles or EPPs (Honys et al., 2000). The identification of the EPPs in the tobacco male gametophyte highlighted the presence of germ cell-like granules in flowering plants. In analogy to the role played by Drosophila melanogaster germinal granules in delivering maternal mRNAs during the initial stages of embryogenesis (Schisa, 2012), in our previous study (Honys et al., 2009), we isolated and characterized the proteome of EPPs and proposed that, during pollen maturation, the storage EPPs represent preloaded complex machinery devoted to mRNA processing, transport, subcellular localization, and protein synthesis (Honys et al., 2009). Discovery of the EPPs also supported the long-hypothesized presence of stored messenger ribonucleoprotein (mRNP) particles in developing tobacco male gametophytes (Honys et al., 2000, 2009). The physiological advantage that the EPPs convey is that, being composed of mRNA sets that are stored and translationally silenced at earlier stages of development, they enable an immediate activation of translation of selected mRNAs during germination of the pollen grain and subsequent pollen tube growth (Honys et al., 2009). This potential role of EPPs particularly resembles that of the growing axons of the human neuronal cells. The directional growth in neurons is facilitated by the transport of sequestered mRNAs by neuronal granules to the synaptic surfaces for translation (for review, see Buchan, 2014). Similar to EPPs, neuronal granules also are preloaded with translational machinery and represent the mediators of nerve cell networking, which deposit transcripts to the growing tip and catalyze their efficient translation, thereby promoting directional growth (Elvira et al., 2006; Hirokawa, 2006). During the progamic phase, an intense molecular dialogue occurs between male and female reproductive tissues (for review, see Vogler et al., 2016) that, among others, involves proteins secreted by growing pollen tubes (Hafidh et al., 2016). In Arabidopsis, a genome-wide study of the in vivo pollen tube translatome revealed that transcripts encoding pollen-secreted proteins are involved in pollen guidance (Lin et al., 2014).
Definition of the pollen sequestrome. A, Quantification of expressed transcripts in total RNA, transcripts expressed in all three fractions, and the overlap of both sets. B, Overall expression signal in all three fractions during pollen development and the progamic phase. C, Relative distribution of the expression signal between subcellular fractions in four stages of pollen development and the progamic phase. D, Average expression signal in each subcellular fraction during pollen development and the progamic phase. E, PCA of transcripts present in all three subcellular fractions and total RNA in MPG. F, PCA of transcripts forming the sequestrome fraction during pollen development and the progamic phase. G, Hierarchical clustering of transcripts present in all three subcellular fractions and in total RNA in mature pollen.
The quantification of expressed genes (Fig. 2A; Supplemental Fig. S1A) confirmed the general trends of general reduction of the total transcriptome complexity during pollen maturation followed by its slight increase after pollen germination. PCA and correlation analyses revealed the developmental shift in the very last period of pollen maturation, since the transcriptomes of immature pollen formed one group different from the transcriptomes of mature pollen and pollen tubes (Supplemental Fig. S3, D and E). In developing pollen, there were smaller differences between the transcriptomes of the subcellular fractions than later, in mature pollen and during the early progamic phase (MPG and PT4). The latter showed higher divergence of translationally active and stored transcript populations, as also confirmed by PCA showing the dynamics of the transcriptomes of the subcellular fractions in the male gametophyte and the relationship of the total and fraction transcriptomes of all six developmental stages (Supplemental Fig. S3C). We also selected five candidate genes with different expression profiles to verify their microarray expression by reverse transcription quantitative PCR (RT-qPCR) in mature pollen in three subcellular fractions (Supplemental Fig. S4).
On the basis of the selected criteria, 37,610 probes (85.9% of 43,803 probes present on the array) had a positive signal in at least one gametophytic or sporophytic data set and, thus, were considered expressed. Of them, 25,034 probes (66.6% of expressed genes) were present in the male gametophyte in at least one developmental stage. Similarly, a previous analysis of the tobacco transcriptome that utilized 40K custom-designed Affymetrix microarrays reported reliable expression of 76% of the probe sets in 19 different tobacco tissues, including leaves and roots (Edwards et al., 2010). The highest number of genes was expressed in early bicellular pollen (Fig. 2A; Supplemental Fig. S1A). This also was the developmental stage with the highest proportion of actively translated transcripts present in the polysomal fraction (Fig. 2, B and C) that gradually decreased later on. On the contrary, the proportion of the sequestrome increased during the final phases of pollen maturation and reached its maximum in mature pollen and 4-h cultivated pollen tubes (Fig. 2, B and C). The dominance of the sequestrome in MPG and PT4 was not only relative but also absolute (Fig. 2D), regardless of the fact that these two developmental stages expressed the lowest number of genes. Of them, the most abundant transcripts were stored in the sequestrome fraction (Supplemental Table S1).
The uniqueness of the total male gametophyte versus sporophyte transcriptomes was already demonstrated (Bokvaj et al., 2014; Supplemental Fig. S3, D and E). We extended the PCA to visualize the relationship of all individual total and fraction transcriptomes in pollen (Fig. 2E) that highlighted the sequestrome as the most unique fraction in mature pollen. The close proximity of all pairs of replicates confirmed the high reproducibility among replicates (Supplemental Fig. S3, B, E, and F). This distribution also was similar at other developmental stages (data not shown). However, the sequestrome dynamics resembled that of total RNA, being formed by a set of transcripts diverting from the sporophytic transcriptomes especially in the late progamic phase (Fig. 2F; Supplemental Fig. S3D). From the observed distribution, we concluded (1) a low degree of posttranscriptional regulation between free mRNPs and polysomes and (2) a great distance between two presumed RNA storage compartments, free mRNPs and EPPs. This suggests a complex and precise regulation of transcript distribution between these two compartments. Free mRNPs probably harbor transcripts that are on their way to becoming associated with polysomes, whereas EPPs were shown to more likely contain long-term-stored transcripts without immediate relation to translation, as already indicated by previously published results (Honys et al., 2009).
The number of proteins associated with individual fractions ranged from 606 to 1,372 (Fig. 4B). The protein quantification in fractions highlighted the reduced translational activity in older pollen tubes (reduction of polysome-associated proteins from 968 to 606) as well as the higher number of proteins associated with the long-term storage of sequestered transcripts that was most apparent in EPPs in PT24. PCA (Fig. 4C) grouped the proteomes of all individual fractions together regardless of the developmental stage. It confirmed that the fraction proteomes throughout development were much more uniform than fraction transcriptomes, suggesting the similar regulatory mechanisms associated with transcript storage. In this respect, EPP and polysomal proteomes were more similar to each other than to the RNP proteome. It likely reflected the presence of the set of ribosomal proteins in both fractions (Honys et al., 2009). Categorization of proteins present in all fractions revealed that the proportion of proteins shared by all three fractions (Fig. 4D) decreased during the progamic phase from 37% (MPG) to 22% (PT24). Accordingly, the proportion of proteins associated with nontranslated transcripts increased gradually, from 13% (MPG) to 33% (PT24) in EPPs and from 16% (MPG) to 26% (PT24) in the RNP fraction. This trend was not followed in the polysomal fraction, where the proportion of fraction-specific proteins decreased from 16% (MPG) to 6% (PT24). On the contrary, proteins forming the polysomal fraction were the most abundant not only among the polysome fraction-exclusive proteins (Fig. 4E) but especially among proteins shared by POL/EPP and POL/EPP/RNP fractions, as documented by their median expression signal (Fig. 4E) as well as the highest and even gradually increasing values in quartiles 2 and 3 of their relative abundance box plot profiles. Accordingly, these polysomal proteins also were by far the most basic, with the median pI value ranging between 9 and 10 (Fig. 4F). Moreover, although the proportion of proteins shared by the POL/EPP and POL/EPP/RNP fractions decreased during the progamic phase, quartiles 2 and 3 increased in abundance, showing that the most abundant proteins formed the core of these fraction proteomes. The proteins shared by all three subcellular fractions also were identified by the highest number of peptides in all three developmental stages (Supplemental Fig. S6). 041b061a72