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Degradome and Secretome of Pollination Drops of Ephedra
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2015 (English)In: The Botanical review, ISSN 0006-8101, E-ISSN 1874-9372, Vol. 81, no 1, 1-27 p.Article in journal (Refereed) Published
Abstract [en]

Although secreted proteins (a secretome) are known to occur in gymnosperm pollination drops, this study shows evidence for the presence of a protein degradome for the first time. A protein degradome is composed of protein and peptide fragments, a product of protein breakdown, whereas a secretome is composed of whole, secreted, and often biologically active extracellular proteins. Harvested Ephedra pollination drops from seven species were pooled either by collection date or, in the case of less abundant sample volumes, by species. Samples were processed by one of two methods: 1. gel electophoresis or by 2. liquid-liquid extraction, followed by chromatographic separation. Processed samples were trypsin-digested and analyzed with a Thermo Scientific LTQ Orbitrap Velos. On average, two-thirds of the detected and characterized proteins found in Ephedra spp. pollination drops were intracellular proteins, such as ubiquitin. The remaining third represent proteins known to be secreted, often involved in apoplastic processes such as defense and carbohydrate-modification, typical of known conifer pollination drop proteins. Characterized proteins detected in our comparative study of Ephedra spp drops ranged from 6 in E. monosperma to 20 in E. foeminea. We propose that the intracellular proteins detected are present as the result of nucellar tissue degeneration during pollination drop formation; previous proteomic investigations of pollination drops were in taxa that lack nucellar degeneration during drop formation Discovery of a degradome in pollination drops is novel and significant in that its presence has biological implications for pollination biology. We predict that degradomes in pollination drops are not restricted to Ephedra, but should also occur in species with nucellar tissue breakdown that coincides with pollination drop formation, such as in cycads and Ginkgo and some Pinaceae. Analysis of several collection dates of E. monosperma shows a large number of proteins that change over the course of the pollination drop secretion period, which suggests that variation in pollination drop contents over time may be important in the pollination biology of Ephdera.

Place, publisher, year, edition, pages
2015. Vol. 81, no 1, 1-27 p.
Keyword [en]
Degradome, Ephedra, Gymnosperm reproduction, Pollination drop, Proteomics, Secretome
National Category
Biological Sciences
Research subject
Plant Systematics
Identifiers
URN: urn:nbn:se:su:diva-116638DOI: 10.1007/s12229-014-9147-xISI: 000350897300001OAI: oai:DiVA.org:su-116638DiVA: diva2:807265
Note

AuthorCount:10;

Available from: 2015-04-23 Created: 2015-04-22 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Evolutionary studies of the Gnetales
Open this publication in new window or tab >>Evolutionary studies of the Gnetales
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Gnetales consist of three distinct genera, Ephedra, Gnetum and Welwitschia with considerable divergence among them regarding morphological, ecological and molecular characters. A longstanding debate of the similarity between the Gnetales and angiosperms and the unresolved seed plant phylogeny intrigues plant scientists to further investigate the evolutionary history of the Gnetales. The presented projects deal with interdisciplinary questions on proteomics, chloroplast genomes, phylogenetic relationships, gross morphology and taxonomy. The thesis aims to summarize general problems encountered in previous studies, and to provide new insights and future perspectives based on the results of completed and ongoing projects.   

In Ephedra, the Mediterranean species E. foeminea has been shown to be entomophilous and it possesses an important phylogenetic status as the sister of the remaining genus. Therefore, the chloroplast genome of E. foeminea was assembled and compared to that previously presented (of the anemophilous Asian species E. equisetina, nested in the core clade of Ephedra). The genome has a quadripartite structure and comprises 118 genes and 109,584 base pairs. A pairwise genome comparison was conducted between E. foeminea and E. equisetina, resulting in the detection of 2,352 variable sites, the obtained data can be used for prospective phylogenetic studies. A proteomic study was also conducted on E. foeminea along with three anemophilous Ephedra species, in order to investigate the biochemistry of the pollination drops. The results show that detected proteins in the pollination drops of Ephedra vary dramatically among species but always occur in very low amounts. The majority of the detected proteins are degradome proteins, i.e., waste products from degrading cells of the nucellus. Some secretome proteins were also found, which are putatively functional, but also these proteins occur in very low amounts. The repeatability of the proteomic studies can, however, be questioned. The sampling methods and proteomic analyses are probably problematic although some suggestions for improvement are provided. Thus I chose to continue with other projects.

In Gnetum, reconstruction of the genus phylogeny and assessments of divergence times of clades were performed using an extensive sampling of ingroup and outgroup accessions. The results show that the South American lineage separated from the remaining genus in the Late Cretaceous. The continued diversification event gave rise to an African lineage and an Asian lineage. The crown age of the Asian clade, which comprises two arborescent species sister to the remaining liaonid species, was estimated to the Cretaceous-Paleogene (K-Pg) boundary. In light of the genus phylogeny and estimated node ages, we suggest that the breakup of Gondwana influenced diversification patterns in Gnetum. Later dispersal events also contributed to the current distribution of Gnetum, and to the phylogenetic patterns within each of the major clades. From my results, it is however clear that taxonomy and species delimitations are poorly defined, and needs to be further studied for all subclades of Gnetum. I have initiated this task by studying the Chinese lianoid clade of Gnetum more in depth. Eleven chloroplast genomes were generated, aligned and compared. Based on the information, four chloroplast markers were designed and applied to further resolve the species relationships with an extensive sampling. The results show, with strong support, that G. parvifolium is sister to all the remaining species of the Chinese linaoid clade. Another five lianoid species are confirmed using both morphological and molecular data, but several names are represented by type material that cannot be considered separate species. Modified keys for identification of male and female plants are presented, based on vegetative and reproductive structures. A subsequent dating analysis indicates that diversification in the Chinese lianoid Gnetum clade took place mainly in the Neogene, during which environmental changes probably facilitated diversification in the lineage.

Place, publisher, year, edition, pages
Stockholm: Department of Ecology, Environment and Plant Sciences, Stockholm University, 2016. 44 p.
National Category
Botany
Research subject
Plant Systematics
Identifiers
urn:nbn:se:su:diva-128439 (URN)978-91-7649-371-7 (ISBN)
Public defence
2016-06-03, Vivi Täckholmsalen (Q-salen), NPQ-huset, Frescativägen 20 A, Stockholm, 13:00 (English)
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Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2016-05-11 Created: 2016-03-28 Last updated: 2017-02-17Bibliographically approved

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