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Proteome of pollination drops in Ephedra (Gnetales)
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.ORCID iD: 0000-0002-8558-5655
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Proteins are important in many physiological and ecological processes. In pollination drops of gymnosperms, proteins take part in a number of pollen-ovule interactions and aid for example in ovule defense. Pollination drops in Ephedra (Gnetales) are secreted in large volumes and are known to be high in sucrose, but their proteomic profile has not been thoroughly studied. In this study, proteomic analysis of pollination drops of Ephedra was carried out based on four species: E. distachya, E. foeminea, E. likiangensis and E. minuta. Pollination drop samples were analyzed using gel electrophoresis (1D SDS-PAGE) and mass spectrometry (LC-MS analysis). Protein profile and putative function of proteins were assessed by searches against a set of proteomic databases. Results indicate that number of proteins as well as their amount was low, and most proteins belong to a degradome, i.e., they are remains of degrading tissue at the apex of the nucellus during formation of the pollination chamber. Variation among species was considerable. Ephedra foeminea, which is sister to all other species, contains more kinds of proteins than other investigated species. The closely related species E. likiangensis and E. minuta differ considerably in protein profiles. In E. distachya, no secretome was found; all detected proteins in this species are degradome. The observed differences between species may be related to pollination biology. Insect pollinated species, such as E. foeminea appear to have more proteins in their pollination drops than wind-pollinated species such as E. distachya. Many things are still uncertain regarding proteomics of pollination drops in Ephedra. Among other things, repeatability of results may be a problem; however, this study clearly shows that the amount of proteins in ephedran pollination drops is very low compared to other studied gymnosperms. Future studies of ovule defense in Ephedra should probably have another focus, and perhaps consider sugar content and carbohydrate metabolism rather than proteomics.

Keyword [en]
pollination drops, Ephedra, proteomics, mass spectrometry, evolution of pollination, ovule defense, carbohydrate metabolism
National Category
Botany
Research subject
Plant Systematics
Identifiers
URN: urn:nbn:se:su:diva-128437OAI: oai:DiVA.org:su-128437DiVA: diva2:915007
Available from: 2016-03-28 Created: 2016-03-28 Last updated: 2016-03-30Bibliographically 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)
Opponent
Supervisors
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: 2016-05-18Bibliographically approved

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