Comparative transcriptomics of seed nourishing tissues: uncovering conserved and divergent pathways in seed plants.

The evolutionary and ecological success of spermatophytes is intrinsically linked to the seed habit, which provides a protective environment for the initial development of the new generation. This environment includes an ephemeral nourishing tissue that supports embryo growth. In gymnosperms this tissue originates from the asexual proliferation of the maternal megagametophyte, while in angiosperms it is a product of fertilization, and is called the endosperm. The emergence of these nourishing tissues is of profound evolutionary value, and they are also food staples for most of the world's population. Here, using Orthofinder to infer orthologue genes among newly generated and previously published datasets, we provide a comparative transcriptomic analysis of seed nourishing tissues from species of several angiosperm clades, including those of early diverging lineages, as well as of one gymnosperm. Our results show that, although the structure and composition of seed nourishing tissues has seen significant divergence along evolution, there are signatures that are conserved throughout the phylogeny. Conversely, we identified processes that are specific to species within the clades studied, and thus illustrate their functional divergence. With this, we aimed to provide a foundation for future studies on the evolutionary history of seed nourishing structures, as well as a resource for gene discovery in future functional studies.

Testing for "snowballing" hybrid incompatibilities in Solanum: impact of ancestral polymorphism and divergence estimates.

Two recent high-profile studies offered empirical evidence for a "snowballing" accumulation of postzygotic incompatibilities in Drosophila and Solanum (tomatoes). Here we present a reanalysis of the Solanum data that is motivated by population genetic principles. Specifically, the high levels of intraspecific nucleotide polymorphism in wild tomato species and presumably large effective population size throughout the divergence history of this clade imply that ancestral polymorphism should be taken into account when evaluating sequence divergence between species. Based on our reanalyses of synonymous-site divergence between the four focal Solanum species and a wide range of ancestral polymorphism, we assessed under which conditions the reported accumulation of seed sterility factors supports the snowball effect. Our results highlight the pivotal impact of levels of ancestral polymorphism and alternate divergence values, and they illustrate that robust tests of the snowball effect in Solanum require genome-wide estimates of divergence.

Endosperm and Seed Transcriptomes Reveal Possible Roles for Small RNA Pathways in Wild Tomato Hybrid Seed Failure.

Crosses between the wild tomato species Solanum peruvianum and Solanum chilense result in hybrid seed failure (HSF), characterized by endosperm misdevelopment and embryo arrest. We previously showed that genomic imprinting, the parent-of-origin-dependent expression of alleles, is perturbed in the hybrid endosperm, with many of the normally paternally expressed genes losing their imprinted status. Here, we report transcriptome-based analyses of gene and small RNA (sRNA) expression levels. We identified 2,295 genes and 387 sRNA clusters as differentially expressed when comparing reciprocal hybrid seed to seeds and endosperms from the two within-species crosses. Our analyses uncovered a pattern of overdominance in endosperm gene expression in both hybrid cross directions, in marked contrast to the patterns of sRNA expression in whole seeds. Intriguingly, patterns of increased gene expression resemble the previously reported increased maternal expression proportions in hybrid endosperms. We identified physical clusters of sRNAs; differentially expressed sRNAs exhibit reduced transcript abundance in hybrid seeds of both cross directions. Moreover, sRNAs map to genes coding for key proteins involved in epigenetic regulation of gene expression, suggesting a regulatory feedback mechanism. We describe examples of genes that appear to be targets of sRNA-mediated gene silencing; in these cases, reduced sRNA abundance is concomitant with increased gene expression in hybrid seeds. Our analyses also show that S. peruvianum dominance impacts gene and sRNA expression in hybrid seeds. Overall, our study indicates roles for sRNA-mediated epigenetic regulation in HSF between closely related wild tomato species.

Laser-Assisted Microdissection of Plant Embryos for Transcriptional Profiling.

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.

The Boechera Genus as a Resource for Apomixis Research.

The genera Boechera (A. Löve et D. Löve) and Arabidopsis, the latter containing the model plant Arabidopsis thaliana, belong to the same clade within the Brassicaceae family. Boechera is the only among the more than 370 genera in the Brassicaceae where apomixis is well documented. Apomixis refers to the asexual reproduction through seed, and a better understanding of the underlying mechanisms has great potential for applications in agriculture. The Boechera genus currently includes 110 species (of which 38 are reported to be triploid and thus apomictic), which are distributed mostly in the North America. The apomictic lineages of Boechera occur at both the diploid and triploid level and show signs of a hybridogenic origin, resulting in a modification of their chromosome structure, as reflected by alloploidy, aneuploidy, substitutions of homeologous chromosomes, and the presence of aberrant chromosomes. In this review, we discuss the advantages of the Boechera genus to study apomixis, consider its modes of reproduction as well as the inheritance and possible mechanisms controlling apomixis. We also consider population genetic aspects and a possible role of hybridization at the origin of apomixis in Boechera. The molecular tools available to study Boechera, such as transformation techniques, laser capture microdissection, analysis of transcriptomes etc. are also discussed. We survey available genome assemblies of Boechera spp. and point out the challenges to assemble the highly heterozygous genomes of apomictic species. Due to these challenges, we argue for the application of an alternative reference-free method for the comparative analysis of such genomes, provide an overview of genomic sequencing data in the genus Boechera suitable for such analysis, and provide examples of its application.

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.