Fay-Wei Li

Evolutionary genomics of seed-free plants

Genomic information is critical to elucidating the evolutionary history and the genetic basis of diverse traits. However, despite over 300 plant genomes having been published, the majority of them were from seed plants and very little is known about seed-free plants. Our group is leading the effort to not only close this large “genome gap”, but also leverage omics data to understand the unique biology of seed-free plants. To date, we have completed the first genomes for ferns, hornworts, and Isoetales, and have more in the pipeline.

Ferns—In 2018, our group led an international collaboration to complete the first genomes of ferns (Li et al., 2018). These genomes, from Azolla and Salvinia, not only provided an exciting glimpse into fern genome structure and evolution, but also clarified the origins of gene families and pathways that coincide with the emergence of seed plants. We have continued to play a lead role in advancing fern genomics. In 2022 we published the genome of the tree fern Alsophila spinulosa (Huang et al., 2022). This genome is notable because Alsophila is homosporous (in contrast to the heterosporous Azolla and Salvinia) and the size, at 6 Gb, is more typical of ferns. We discovered a remarkably preserved synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago. Such a slow rate of genome fractionation is unprecedented and might explain the large genome sizes found in ferns. In parallel, we have also been working closely with Open Green Genomics (a JGI-funded project) and aim to have four more fern genomes completed by the end of 2022.

Lycophytes—There are three extant and deeply diverged lineages in lycophytes: Selaginellales, Isoetales, and Lycopodiales. For years, genomic data had only been available for Selaginella in Selaginellales. Recently, we made important progress in lycophyte genomics with the completion of Isoetes taiwanensis (Isoetales) genome (Wickell et al., 2021). Our interest in I. taiwanensis is primarily in its Crassulacean acid metabolism (CAM) photosynthesis, which is unusual given the aquatic lifestyle. From our time-course RNA-sequencing data, we found that I. taiwanensis may have recruited the lesser-known “bacterial-type” phosphoenolpyruvate (PEP) carboxylase, along with the “plant-type” exclusively used in other CAM and C4 plants for carboxylation of PEP. Our findings suggest the existence of more evolutionary paths to CAM than previously recognized. For the remaining order Lycopodiales, we have finalized the genomes of Diphasiastrum complanatum and Huperzia asiatica, which, together with those of Selaginella and Isoetes, will give us a much more holistic view of the lycophyte genomic landscape.

Hornworts—Hornworts are one of the three lineages of bryophytes, whose phylogenetic position among land plants has been highly debated. In 2020 we published the first hornwort genomes from two Anthoceros species (Li et al., 2020). Our genomic analyses provided support for the monophyly of bryophytes and help address several long-standing questions on early land plant evolution. Since then, we have massively expanded the sampling, with 10 new high-quality genomes from every hornwort family—hornworts are now the best sampled plant phylum to date. This genomic framework is paving the way to establish hornworts as a new model lineage.

Hornworts as a new model system

Hornworts are distinct in many aspects. Phylogenetically, hornworts are one of the major plant lineages that diverged from other extant land plants over 480 million years ago. As such, hornworts are of paramount importance to retrace plant evolutionary history. Physiologically, hornworts have endophytic symbiosis with mycorrhizal fungi and N2-fixing cyanobacteria, pyrenoids for concentrating CO2, a chimeric photoreceptor, and U/V sex chromosomes. Developmentally, hornwort sporophytes are unique in having a basal semi-indeterminate meristem and stomata that are perpetually open. Research on hornworts can therefore bring novel insights into a wide range of biological disciplines, such as plant evolution, evo-devo, plant-microbe interactions, and carbon fixation. In order to facilitate hornwort research, we have been building several key resources and foundational tool:

Hornwort-cyanobacteria symbiosis

Plant symbiosis with nitrogen-fixing cyanobacteria is a unique form of mutualistic association that has independently evolved in diverse lineages including a few species of bryophytes, ferns, cycads, and one small genus of flowering plants. Compared to other nitrogen-fixing microbes, cyanobacteria are generally less dependent on the plant host, and therefore could be an ideal partner for engineering symbiotic nitrogen fixation into crop plants. However, our current understanding of plant-cyanobacteria symbioses is rudimentary. The phylogenetic diversity of cyanobionts has been largely unexplored, and very few studies have investigated variation in the symbiotic interaction. In addition, most genetic research has solely focused on the model cyanobiont Nostoc punctiforme, and the plant genes involved in symbiosis remain unknown.

Carbon-concentrating mechanism in hornworts

To enable more efficient photosynthesis, certain organisms have evolved biophysical carbon concentrating mechanisms (CCM) that partition Rubisco (nature’s carbon fixing enzyme) and CO2 into a dedicated subcellular compartment. Pyrenoids are an example of such compartments—organelles comprised of an interconnected matrix of aggregated Rubisco that are liquid-liquid phase separated from the stroma. The green alga Chlamydomonas reinhardtii has been the traditional model to study pyrenoid-based CCMs with the hope of installing a similar mechanism into crops to enhance yield. On the other hand, hornworts are the only land plants that have a pyrenoid-based CCM. Owing to their much closer relationship to crop plants, lessons learned from hornworts might have higher translational potential than those from Chlamydomonas. Another advantage of the hornwort system is that CCMs have been repeatedly gained and lost over the past 50 million years in the evolution of this plant group, offering a unique phylogenetic replication for comparative studies.

To date, no hornwort CCM component has been characterized and nothing is known about what mediates Rubisco aggregation in hornwort pyrenoids. Together with Laura Gunn’s group at Cornell University, we recently secured a collaborative NSF grant to study the hornwort CCM. The plan is to use a three-pronged approach—combining comparative genomics, RNA-seq, and proteomics—to identify putative CCM components, leveraging repeated pyrenoid-present/absent transitions to narrow in on candidates. Ultimately, we aim to use a constructionist approach to characterize interactions between Rubisco and recombinantly-produced putative linker proteins in vitro, and assess the potential for Rubisco phase separation. The outcomes of this research should provide clues as to how a biophysical CCM can be assembled in land plants.

Supported by NSF MCB Cellular Dynamics and Function | From phylogeny to biomolecules: a cross-scale approach to understand the making of a unique carbon-concentrating mechanism in hornworts. PI: Fay-Wei Li, Laura Gunn (Cornell Plant Biology)