Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

Abstract

BackgroundAs in animals, cell–cell communication plays a pivotal role in male–female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective.ResultsWe performed genome-wide quantitative liquid chromatography–tandem mass spectrometry analysis of a pistil-stimulated pollen tube secretome and identified 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube secretome is dominated by proteins that are secreted unconventionally, representing 57 % of the total secretome. In support, we show that an unconventionally secreted protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discovered that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes, as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that translationally controlled tumor protein-knockdown Arabidopsis thaliana plants produce pollen tubes that navigate poorly to the target ovule and that the mutant allele is poorly transmitted through the male. Further, we show that regulators of the endoplasmic reticulum–trans-Golgi network protein secretory pathway control secretion of Nicotiana tabacum Pollen tube-secreted cysteine-rich protein 2 and Lorelei-like GPI-anchor protein 3 and that a regulator of endoplasmic reticulum–trans-Golgi protein translocation is essential for pollen tube growth, pollen tube guidance and ovule-targeting competence.ConclusionsThis work, the first study on the pollen tube secretome, identifies novel genome-wide pollen tube-secreted proteins with potential functions in pollen tube guidance towards ovules for sexual reproduction. Functional analysis highlights a potential mechanism for unconventional secretion of pollen tube proteins and reveals likely regulators of conventional pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggests exosome secretion is a possible mechanism for cell–cell communication between the pollen tube and female reproductive cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0928-x) contains supplementary material, which is available to authorized users.

The Molecular Dialog between Flowering Plant Reproductive Partners Defined by SNP-Informed RNA-Sequencing

“…Interestingly, genes that require MYB transcription factors for expression in the pollen tube are enriched for secreted proteins including two alpha carbonic anhydrases ( Figures 4O to 4T), 11 endopeptidases, 22/28 thionins in the CRP2460 gene family (Figure 6), 19/98 S-protein homologs (Ride et al, 1999), two cysteine-rich secretory, antigen 5, and pathogenesisrelated 1 proteins (CAP; AT3G09590 and AT5G02730), and four rapid alkalinization factors (AT2G32835, AT2G32788, AT2G32785, and AT1G60815; Supplemental Data Sets 8 and 9). Interestingly, a comparison between the tobacco pollen tube and A. thaliana MYB-dependent pollen tube genes revealed significant overlap both in terms of specific gene homologs and for functional gene categories (Hafidh et al, 2016). These findings suggest a core group of proteins secreted by pollen tubes of dicot species that could be important for sperm release.…”

“…The thioninlike family of small cysteine-rich proteins provides a striking example: A single MYB-dependent member was identified by microarray analysis (Leydon et al, 2013), while RNA-seq analysis identified 24 family members whose expression was significantly decreased in myb triple-mutant pollinations compared with the wild type ( Figure 6). MYB-dependent secreted proteins were of particular interest because recent analysis documented the rich diversity of the tobacco (Nicotiana tabacum) pollen tube secretome (Hafidh et al, 2016) and suggested that the pollen tube and the synergid cell (Punwani et al, 2007;Liu et al, 2015) both evolved highly active secretory systems. Interestingly, genes that require MYB transcription factors for expression in the pollen tube are enriched for secreted proteins including two alpha carbonic anhydrases ( Figures 4O to 4T), 11 endopeptidases, 22/28 thionins in the CRP2460 gene family (Figure 6), 19/98 S-protein homologs (Ride et al, 1999), two cysteine-rich secretory, antigen 5, and pathogenesisrelated 1 proteins (CAP; AT3G09590 and AT5G02730), and four rapid alkalinization factors (AT2G32835, AT2G32788, AT2G32785, and AT1G60815; Supplemental Data Sets 8 and 9).…”

Secretomes of Buckwheat Suspension Cultures With Different Morphogenic Activity

“…Кроме того, среди белков, не имеющих N-концевого сигнального пептида, были выявлены антиоксидантные ферменты (глутатион-S-трансферазы, каталаза, глутатионредуктаза), шапероны (белки теплового шока, 14-3-3 белки, калретикулин, кальмодулин), а также сериновые и аспарагиновые эндопептидазы, ингибиторы сериновых протеаз. Ортологи этих белков были обнаружены в секретомах других растений [Pechanova et al, 2010, Gupta et al, 2011Hafidh et al, 2016]. Полученные результаты указывают на вклад неклассической секреции белков в формирование секретома суспензионных культур F. tataricum.…”

“…В секретоме пыльцевых трубок [Hafidh et al, 2016] и в экссудатах кончиков корней [Wen et al, 2007] их содержание достигает 80%. Большее содержание белков без сигнального пептида в МСК по сравнению с НСК может свидетельствовать о том, что процессы дифференцировки в растениях (в случае МСК -формирование проэмбриональных клеточных комплексов-ПЭКК), так же как и в животных, сопряжены с неклассической секрецией белков.…”

Gametophytic Pollen Tube Guidance: Attractant Peptides, Gametic Controls, and Receptors

“…The male gametophyte consists of three cells with two cell types: two small sperm cells and one vegetative cell (tube cell) that encloses the two sperm cells within its endocytic membrane (Borg et al, 2009;McCue et al, 2011;Hafidh et al, 2016b). In flowering plants, nonmotile sperm cells are delivered by the pollen tube, a tubular cell structure that grows from the vegetative cell of pollen and navigates to the female gametophyte, where the egg cell resides (Fig.…”