Bouatta, A. M. | bioRxiv Science

bioRxiv2024

Polarised subcellular activation of ROPs by specific ROPGEFs drives pollen germination in Arabidopsis thaliana

During plant fertilisation, excess male gametes compete for a limited number of female gametes. The dormant male gametophyte, encapsulated in the pollen grain, consists of two sperm cells enclosed in a vegetative cell. After reaching the stigma of a compatible flower, quick and efficient germination of the vegetative cell to a tip-growing pollen tube is crucial to ensure fertilisation success. RHO OF PLANTS (ROP) signalling and their activating ROP GUANINE NUCLEOTIDE EXCHANGE FACTORS (ROPGEFs) are essential for initiating polar growth processes in multiple cell types. However, which ROPGEFs activate pollen germination is unknown. We investigated the role of ROPGEFs in initiating pollen germination and the required cell polarity establishment. Of the five pollen-expressed ROPGEFs, we found that GEF8, GEF9, and GEF12 are required for pollen germination and male fertilisation success, as gef8;gef9;gef12 triple mutants showed almost complete loss of pollen germination in vitro and had a reduced allele transmission rate. Live cell imaging and spatiotemporal analysis of subcellular protein distribution showed that GEF8 and GEF9, but not GEF12, displayed transient polar protein accumulations at the future site of pollen germination minutes before pollen germination, demonstrating specific roles for GEF8 and GEF9 during the initiation of pollen germination. Furthermore, this novel GEF accumulation appears in a biphasic temporal manner and can shift its location. We showed that the C-terminal domain of GEF8 and GEF9 confers this protein accumulation and demonstrated that GEFs locally activate ROPs and alter Ca2+ signalling, which is required for pollen tube germination. We demonstrated that GEFs do not act redundantly during pollen germination and described for the first time a polar domain with spatiotemporal flexibility, which is crucial for the de novo establishment of a polar growth domain within a cell and, thus, for pollen function and fertilisation success.

plant biology↗

bioRxiv2020

RAD54 is essential for RAD51-mediated repair of meiotic DSB in Arabidopsis.

An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates homology search and strand invasion activity of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Absence of RAD54 in dmc1 mutants leads to meiotic chromosomal fragmentation. The action of RAD54 in meiotic RAD51 activity is thus downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination. Author SummaryHomologous recombination is a universal pathway which repairs broken DNA molecules through the use of homologous DNA templates. It is both essential for maintenance of genome stability and for the generation of genetic diversity through sexual reproduction. A central step of the homologous recombination process is the search for and invasion of a homologous intact DNA sequence that will be used as template. This key step is catalysed by the RAD51 recombinase in somatic cells and RAD51 and DMC1 in meiotic cells, assisted by a number of associated factors. Among these, the chromatin-remodelling protein RAD54 is a required cofactor for RAD51 in mitotic cells. Understanding of its role during meiotic recombination however remains elusive. We show here that RAD54 is required for repair of meiotic double strand breaks by RAD51 in the plant Arabidopsis thaliana, and this function is downstream of the meiotic role of RAD51 in supporting the activity of DMC1. These results provide new insights into the regulation of the central step of homologous recombination in plants and very probably also other multicellular eukaryotes.

genetics↗