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Building and bypassing plant polyspermy blocks

Periodic Reporting for period 4 - bi-BLOCK (Building and bypassing plant polyspermy blocks)

Reporting period: 2020-03-01 to 2022-01-31

It was considered an inviolable principle that sexually reproducing organisms have no more than two parents and fertilization of an egg by multiple sperm (polyspermy) is lethal in many eukaryotes. As neither the public, nor scientists or breeders were aware that plants can have three parents three-parent crosses were not considered as a research topic or breeding tool. We established a high-throughput polyspermy detection assays, which uncovered that supernumerary sperm fusion does occur in planta and can generate viable polyploid offspring (1). Moreover, polyspermy can give rise to seedlings with one mother and two fathers, challenging the bi-organismal concept of parentage. The economic and societal impact of our discovery is gaining pivotal importance in light of rapid worldwide changes, including global warming and a growing world population, because three parent crosses can speed up breeding processes. We also show that three-parent crosses can bypass hybridization barriers (2). This can revolutionize the ability of breeders to develop improved crops in providing a tool to mine beneficial gene variants of currently incompatible species and to substantially shorten time to market of climate-adapted seed varieties. In addition, we show that the endopeptidases ECS1 and ECS2 regulate offspring genome size via polyspermy and haploid induction. ECS1 and ECS2 are the first molecular factors demonstrated to affect polyspermy in plants (3).
Identification of 3-parent plants in the model organism Arabidopsis thaliana
We established a high-throughput polyspermy detection assays, which uncovered that supernumerary sperm fusion does occur in planta and can generate viable polyploid offspring. Moreover, polyspermy can give rise to seedlings with one mother and two fathers, challenging the bi-organismal concept of parentage. Our data implies that every Arabidopsis thaliana plant can give rise to about 5 polyspermy-induced polyploid plants under ideal growth conditions. The polyspermy derived triploids are taller and produce bigger organs than plants resulting from a regular monospermic fertilization. In addition, we demonstrate the hybridization potential of polyspermy by instantly combining three different Arabidopsis accessions in one zygote. Our results provide direct evidence for polyspermy as a route towards polyploidy, which is considered a major plant speciation mechanism.
Demonstration that 3-parent crosses can bypass a dominant hybridization barrier
The so-called triploid block is an established and widely distributed postzygotic hybridization barrier of the endosperm. Considering its fatal consequence, it has been suggested that there are ways to overcome this barrier. We established a polyspermy detection assay that allows to identify and characterize developing embryos resulting from supernumerary sperm fusion. With this tool we were able to show that most polyspermy-derived plants develop from seeds resulting from selective egg cell polyploidization. In those seeds, supernumerary paternal copies are only transmitted to the embryo, thereby bypassing the triploid block of the endosperm. Our results expand previous polyploidization concepts, which state that the increase in genome copies is caused by infrequent meiotic or mitotic defects. In fact, the currently favored route towards polyploid plants involves unreduced male gametes; however, this scenario introduces supernumerary paternal copies also to the endosperm. Plant polyploidization via polyspermy, by contrast, often affects the embryo-derived seed fraction only and hence can bypass the triploid block of the nourishing tissue.
ECS1 and ECS2 regulate polyspermy and suppress the formation of haploid plants by promoting double fertilization
With ECS1 and ECS2 we identified the first molecular components to regulate polyspermy frequencies. The genes code for endopeptidases and loss-of-function is associated supernumerary pollen tube attraction and an increased polyspermy frequency. Intriguingly, ecs1 ecs2 double mutants also induce haploidy, which is an agronomically highly relevant trait. The results that we gained are conflicting with a recent publication by Yu et al. in the journal "Nature" ( where the authors also observed supernumerary pollen tube attraction but no reproductive defect.

We have teamed up with KWS Saat SE & Co. KGaA (KWS), one of the world's leading plant breeding companies, and the potato breeder Aardevo to transfer the three-parent-breeding technologies to crop plants. Exploitation and dissemination of our results is supported by an ERC Proof of Concept Grant and an EIC transition grant.
Our results got reviewed in leading research journals and broadcasted by 3Sat, Sat1, and Radio Bremen, as well as a podcast by the Naked Scientists.

1. Nakel, T., Tekleyohans, D.G. Mao, Y., Fuchert, G., Vo, D., Gross-Hardt, R. (2017): Triparental plants provide direct evidence for polyspermy induced polyploidy. Nature Communication doi: 10.1038/s41467-017-01044-y
2. Mao, Y., Gabel, A., Nakel, T., Viehoever, P., Baum, T., Tekleyohans, D.G. Vo, D., Grosse, I., Gross-Hardt, R. (2020): Selective egg cell polyspermy bypasses the triploid block. eLife. doi: 10.7554/eLife.52976.
3. Mao, Y, Nakel, T, Erbasol Serbes, I, Tekleyohans, DG, Joshi, S, Baum, T, Groß-Hardt, R (2022) Preprint. bioRxiv
We have established a High-throughput-polyspermy-detection assay (HIPOD), which allows for the first time to detect plants of polyspermic origin. We, in addition, have adapted the technology to detect seeds containing triparental embryos. We have established three parent crosses as a breeding tool and have filed a patent for this new technology (EP 17 748 664.4 US 16/318,830, CN201780044659.9).
To further promote exploitation and dissemination of our results, we successfully applied for an ERC Proof of Concept Grant and, together with the breeders KWS Saat SE & Co. KGaA (KWS) and Aardevo, for an EIC transition grant.