A comprehensive tissue type-specific transcriptional study identifies 45 genes consistently differentially expressed during Boechera sexual as compared with apomictic germline specification, suggesting their relevance for apomixis.

Plant sexual and asexual reproduction through seeds (apomixis) is tightly controlled by complex gene regulatory programs. These are to date not fully understood. Recent findings suggest that RNA helicases are required for plant germline development. This resembles their crucial roles in animals, where they are involved in controlling gene activity and the maintenance of genome integrity. Here we identified novel roles of Arabidopsis RH17 during reproductive development. Interestingly, RH17 is involved in repression of reproductive fate and of elements of seed development in the absence of fertilization. In lines carrying a mutant rh17 allele, development of supernumerary reproductive cell lineages in the female flower tissues (ovules) was observed, occasionally leading to formation of two embryos per seed. Furthermore, seed coat and putatively also endosperm development frequently initiated autonomously. Such induction of several features phenocopying distinct elements of apomixis by a single mutation is rather exceptional and suggests that RH17 acts in regulatory control of plant reproductive development. In longer terms, an in depth understanding of its action might provide a basis for harnessing for agricultural applications.

During sexual reproduction of higher plants, seed formation is initiated by double fertilization of egg and central cell. In contrast, pseudogamous apomicts form embryos asexually by parthenogenesis of the egg, but initiation of endosperm development still depends on central cell fertilization. It can be envisioned that these differences are determined during gametogenesis and specification of gametophytic cells. To deepen the understanding of the transcriptional basis underlying sexual and apomictic reproduction, we applied tissue type-specific RNA-Seq. We compared expression in reproductive tissues of different Boechera accessions at distinct developmental stages. This confirmed previous evidence for an enrichment of RNA helicases at onset of reproductive development. We further identified a small number of members of this gene family as differentially expressed in female reproductive ovule tissues harbouring mature gametophytes from apomictic and sexual accessions. This included homologs of A. thaliana FASCIATED STEM 4 (FAS4) and of ENHANCED SILENCING PHENOTYPE 3 (ESP3), which have previously been identified as potential candidates for gametogenesis and apomixis, respectively. Unlike in A. thaliana, for either of them additional homologs or copies of related genes are present in Boechera, indicating complex evolutionary histories. As the expression patterns implied potential roles of FAS4 during gametogenesis, we first studied A. thaliana lines carrying mutant alleles. Indeed, we observed defects during male and female gametogenesis and severely reduced transmission efficiencies through both parents. In conclusion, our study identifies FAS4 as crucial for plant reproduction and suggests the potential for sub-functionalization of additional homologous genes in Boechera to shape reproductive development.

Author contributionAS conceived the project. AS and LZ planned the experiments. LZ conducted the experiments with support of DI and AS. LZ conducted the data analysis. LZ, AS, and DI interpreted the data.LZ and AS wrote the manuscript. All authors approved the manuscript. One sentence summaryA comprehensive tissue type-specific transcriptional analysis using laser-assisted microdissection combined with RNA-Seq identifies 45 genes consistently differentially expressed during germline specification in different sexual versus apomictic Boechera accessions, indicating roles of protein degradation related to cell cycle, transcriptional and posttranscriptional regulatory processes, and stress response for apomixis. ABSTRACTGermline specification is the first step during sexual and apomictic plant reproduction. This takes place in a specialized domain of the reproductive flower tissues, the nucellus of the ovule.In each case, a sporophytic cell is determined to initiate germline development. These cells, the megaspore mother cell (MMC) or apomictic initial cell (AIC) in sexual plants and apomicts, respectively, differ in their developmental fate. While the MMC undergoes meiosis, the AIC aborts or omits meiosis to form the female gametophyte. Although these distinct developmental processes have long been described, little is known about the gene regulatory basis involved.To elucidate gene regulatory networks underlying sexual and apomictic germline specification, we conducted tissue-specific transcriptional profiling using laser-assisted microdissection and RNA-Seq. We compared the transcriptomes of the nucellar tissues harbouring the MMC or AIC between different accessions of Boechera. The six accessions we used represented four species and two ploidy levels, allowing us to distinguish between differences in gene expression caused by the genetic background or the reproductive mode.Comparative data analysis revealed widely overlapping gene expression patterns in apomictic versus sexual Boechera accessions. Nevertheless, 45 significantly differentially expressed genes were identified, which potentially play a role for determination of sexual versus apomictic reproductive mode. Interestingly, based on annotations, these include F-box proteins and E3 ligases that might relate to genes previously described as regulators important for sexual or apomictic reproduction. Thus, our findings provide new insight into the transcriptional basis of sexual and apomictic germline specification.