Identification and Exploration of Pollen Tube Small Proteins Encoded by Pollination-Induced Transcripts

Abstract

Pollination is composed of cell-cell communication and complicated signaling cascades that regulate pollen tube growth and guidance toward the ovules for double fertilization, and is critical for successful sexual reproduction. Exploring expression profiles of in vivo grown pollen tubes is important. Nevertheless, it is difficult to obtain accessible pollen tubes for profiling studies in most model plants. By taking advantage of the hollow styles of lily (Lilium longiflorum), in vivo pollen tubes harvested from pollinated styles which had been cut open were used here to study their protein and transcript profiles. Pollination quantitatively and qualitatively altered the total protein composition of elongating pollen tubes. cDNAs generated and amplified from total RNAs of 24 h in vivo grown and 12 h in vitro cultured pollen tubes were used for suppression subtractive hybridization analyses and preparation of home-made array chips. Microarray analyses conducted with different probe sets revealed 16 transcripts specifically present and/or enriched in in vivo pollen tubes. Reverse transcription-PCR (RT-PCR), in situ hybridization and Northern blotting were applied to validate their unique pollination-induced expression features. Interestingly, several transcripts were simultaneously detected on the stylar transmitting tract epidermis, where in vivo pollen tubes tightly adhered during pollination. Their deduced amino acid sequences showed that most of them encoded small proteins and could be classified into several families. Transient assay revealed filament-like structures decorated by these proteins and one probably localized in the generative cell. These small peptides might be critical for pollen tube growth during pollination, and further exploration of their biological functions and mechanisms of action are of great interest.

De novo sequencing and analysis of the lily pollen transcriptome: an open access data source for an orphan plant species

“…To name just a few examples, measurements on endogenous ion currents, cytosolic ion gradients, ion channels, turgor pressure or secretion started with lily pollen tubes (Benkert et al 1997;Holdaway-Clarke et al 1997;Messerli et al 1999;Nobiling and Reiss 1987;Obermeyer and Kolb 1993;Obermeyer and Weisenseel 1991;vanderWoude et al 1971;Weisenseel et al 1975). Although this model pollen contributed much to our understanding of physiological processes during pollen tube growth, only a few comprehensive studies of the lily pollen transcriptome were performed, so far (Huang et al 2011(Huang et al , 2006. But from both studies only a few, namely 100 clones and 4 clones, respectively, were sequenced and deposited at GenBank data base.…”

“…But from both studies only a few, namely 100 clones and 4 clones, respectively, were sequenced and deposited at GenBank data base. With the optimisation of particle bombardment conditions (Wang and Jiang 2011) and the use of reliable promoter sequences (Hamilton et al 1992), lily pollen can now be transiently transformed and the localisation of identified proteins can be visualised as shown exemplarily for lily pollen Rop GTPases (Huang et al 2011). In addition, 'knock-out' pollen using anti-sense RNA (Moutinho et al 2001a;Potocky et al 2007) may be generated to investigate the physiological function of the respective protein.…”

“…To utilise these experimental advantages of a non-sequenced pollen species in future studies, more sequence data are required for the identification of molecular processes during germination and tube growth. So far, sequence information on L. longiflorum is very scarce (<1,000 nucleotide entries in NCBI data base, June 2014), and as a consequence no microarrays are commercially available for L. longiflorum, and only a few transcriptome studies on generative lily pollen cells (Okada et al 2006(Okada et al , 2007 and entire pollen grains (Huang et al 2011(Huang et al , 2006 were published until now. To obtain a first overview on the lily pollen transcriptome, a next generation sequencing project was designed for the non-sequenced organism lily pollen to identify as many transcripts as possible based on recommendations for non-model species (Strickler et al 2012;Ward et al 2012).…”

Profiling of Translatomes of in Vivo–Grown Pollen Tubes Reveals Genes with Roles in Micropylar Guidance during Pollination in Arabidopsis

“…In addition, the pollen tube must dynamically adjust its growth pattern based on complicated cell-to-cell interactions with the female pistil and on its rapid and long journey toward the ovule. In lily pollen tubes, this task requires dynamic biosynthesis of mRNA transcripts and proteins during in vivo growth (Huang et al, 2011). To investigate the transcriptome changes of pollinated pollen tubes, a semi-in vivo approach was used, identifying 383 genes enriched in pollen tubes emerging from cut pistils compared with in vitro-cultured ones (Qin et al, 2009).…”

“…To investigate the transcriptome changes of pollinated pollen tubes, a semi-in vivo approach was used, identifying 383 genes enriched in pollen tubes emerging from cut pistils compared with in vitro-cultured ones (Qin et al, 2009). Moreover, by taking advantage of the hollow styles of Easter lilies (L. longiflorum cv Avita and cv Snow Queen), we were able to collect in vivo-grown pollen tubes to identify novel pollen tube transcripts and proteins induced by pollination (Huang et al, 2011).…”