Investigating Possible Sex Determination Genes HD-ZIP and TCP1 in Hornworts

First dual-sex hornwort genomes probe sex chromosome evolution in a haploid system

Hornworts (Anthocerophyta) are a group of seed-free, nonvascular plants known for their horn-shaped sporophyte generation. Shared only with some other seed-free plants and fungi, their unusual haploid-dominant life cycle sees the haploid gametophyte phase as an independent organism and the diploid sporophyte phase as an outgrowth dependent on the gametophyte. Dioicy, or separate sexes, has evolved independently in about 40% of extant species, every time with a UV sex chromosome system. Theoretical models predict that hornworts’ haploid-dominant lifecycle minimizes genetic “shielding” that typically occurs in diploid heterogametic (XY/ZW) systems, thereby improving selection’s ability to purge deleterious mutations on these UV chromosomes and perhaps accelerating sex chromosome differentiation toward sex-specific functions. Therefore, we ask: do hornwort sex chromosomes exhibit either sex-specific differentiation or degradation when compared to heterogametic systems? Here, we assemble the first full male and female genomes of two hornwort species and employ annotation summaries to demonstrate that, though these sex chromosomes are degraded relative to autosomes, degradation is roughly equivalent between sexes. Further, synteny analysis suggests considerable divergence between independently evolved sex chromosome systems.

Coming from a school with a very small plant biology curriculum, I was awed by the breadth and depth of plant science at BTI and Cornell. The opportunity to meet professionals who have devoted their lives to the taxa that fascinate me has galvanized my interest in graduate school. For example, the commitment of my mentor, Peter Schafran, to hornworts and quillwortd—obscure clades of seed-free plants—inspires me to discover my own fervor for natural history because I find few things more meaningful. I’m grateful for the wet lab, computational, and field work I was privileged to partake in, for these helped me discover the obstacles between where I am now and where I wish to be in my career.

Examining Patterns of Dioicy Across Hornwort Phylogeny Using U/V Sex Chromosomes

Sexual reproduction is in every clade within eukaryotes and is essential to life on earth. However, little is known about how it evolved, the development of separate sexes, and what genes trigger this development. The hornworts, a lineage of bryophytes, is one of the deepest diverging clades in land plants. Recent studies have found that 40% of hornwort species are dioicous, with several independent transitions from monoicy to dioicy across the phylogeny. The genes responsible for this evolution are a mystery. To address this, this study compared genomes of male and female individuals from three species of three different families to determine whether similar genes are found in independently formed sex-determining regions. If certain genes have become sex-linked repeatedly, it would suggest these genes have a role in the evolution of dioicy in hornworts. The study began with DNA and RNA extraction for short-read Illumina sequencing of the three species’ genomes. After sequencing, genomes were assembled and aligned to their reference genomes, which led to preliminary data regarding the structure and location of the species sex chromosomes. The locations of the putative sex chromosomes, pseudoautosomal regions, and sex determining regions were found in Leiosporoceros and Phaeomegoceros. In Phymatoceros, the sex chromosome and the sex determining region has been theorized to be found as well. Genes were annotated using RNA data, and homologs to the reference sex were identified. These findings give a foundation for further research to examine whether similar genes are found in independently formed sex-determining regions.

This summer was incredibly valuable in honing my skills as a scientist. Under my mentor’s guidance, I was able to learn skills about troubleshooting difficult plant chemistry and bioinformatic problems while working with whole-genome level datasets. These skills are essential for scientists, and I feel as though I will come out of this summer a much better scientist than when I came in. I feel ready and inspired to go to graduate school and feel empowered to tackle much bigger scientific questions than I ever thought possible. This internship has set me up for a lifetime of learning and a trajectory of scientific curiosity.

“Biological Ballistics”: Optimizing Biolistic Transformation in Hornwort Model Species Anthoceros agrestis

Upon transitioning from an aquatic to a terrestrial environment approximately 500 million years ago, land plants diverged into two major groups, vascular plants and bryophytes. Hornworts, along with liverworts and mosses, make up the bryophytes, which are sister to vascular plants. Despite the insights hornworts could provide to better understanding early land plant evolution, much about the biology of hornworts remains to be studied. It is known that hornworts are the only land plants to contain pyrenoids within their chloroplasts, which function to concentrate CO2 in the nearby environment of RuBisCo, leading to more efficient photosynthesis by reducing the rate of photorespiration. Hornworts also have a symbiotic relationship with cyanobacteria, which fix atmospheric nitrogen for the plant. Integrating these features into crops could have implications for increasing yields and decreasing fertilizer dependency. In order to study the genes responsible for these features, a biolistic transformation method was employed for hornwort model species Anthoceros agrestis. While the current protocol has been used to successfully transform these plants, the efficiency remains low. Four variables in the current biolistic procedure were tested in order to optimize the current protocol: the amount of DNA used, the shooting distance, the DNA precipitation method, and the days post-homogenization. The results of this project aid in the understanding of which factors have the largest influence on the successful transformation rate and could be useful for studying pyrenoids and cyanobacteria symbiosis in the future.

This internship has been a great introduction into what it might be like to conduct research in the realm of plant genetic engineering full-time. I’ve gained experience with biolistic transformation, conducting minipreps, PCR, and using golden gate cloning that will be useful in the future as I pursue graduate school and a career in this field. I’ve learned how to identify problems, adapt procedures, and to remain patient and optimistic when results end up less than ideal while running experiments. Further, I now have a better understanding of the expectations of professional research and how to effectively present my results to various audiences. I’m very grateful to have had this internship opportunity this summer and am excited to see where these new research skills can take me.

“Developing Griffithsia monilis as a model for the study and kinetic optimization of rubisco”

Project Summary:

Rubisco, a vital but inefficient carbon-fixing enzyme, is the limiting factor in green plant growth. The rubisco found in red algae Griffithsia monilis exhibits superior kinetic properties to that of many plants, making it a potential target for genetic modification of green plants. Successful assembly of Griffithsia rubisco has not been observed in green plants, possibly due to unmet chaperone and assembly factor requirements. Studying the genome and proteome of the Griffithsia chloroplast could provide valuable insight into these assembly requirements. We sequenced the Griffthsia genome using Oxford Nanopore MinION technology and assembled organellar genomes from this data, as well as previously obtained Illumina data, using GetOrganelle and Flye. Genomes were corrected with Ratatosk and NextPolish and annotated with known and unknown genes. After bacterial contamination was discovered and confirmed in multiple liquid cultures of the algae, we tested several antibiotics in order to create an antibiotic cocktail to remove bacteria without harming the algae. In addition, we tested two different protocols for the isolation of chloroplasts, one using a sucrose gradient and another using a Percoll gradient for differential centrifugation, as well as testing what methods of lysing algal cells produced the most purified chloroplasts. In order to use Griffithsia to optimize photosynthesis in other plants, we have isolated its organellar genomes and determined protocols to purify its chloroplasts, allowing for the study of red algal rubisco assembly requirements.

My Experience:

Working in research full-time has helped me gain experience and confidence in the lab, as well as in my long-term goals. I assisted with multiple parts of a complex, ongoing project between the Li and Gunn labs, which allowed me to learn a variety of new lab and bioinformatic techniques. I worked with several different members of my lab and was able to learn from their individual interests and areas of expertise. This collaborative experience will be valuable as I continue to work with mentors and peers in my undergraduate and graduate education. In addition, I gained a new appreciation for and understanding of plant biology. I’m glad I was able to contribute to these meaningful projects, and I leave with many new questions about plant biology that I’d love the opportunity to study again someday.

“Exploring the Biolistic Gene Transfer Approach for the Model Hornwort Anthoceros agrestis“

Project Summary:

Approximately 500 million years ago the first algae washed ashore and began the land plant evolution. The first land plants were nonvascular plants called bryophytes. The bryophyte clade encompasses mosses, liverworts, and hornworts. Although research on mosses and liverworts is growing abundantly, little research focuses on hornworts. Anthoceros agrestis is a model species for hornworts that can be used to understand the unique characteristics of hornworts in addition to the mechanisms of evolution in land plants. Pyrenoids are one of these characteristics and have the potential to be implemented into crop plants. Pyrenoids are compartments within algae and hornwort chloroplasts that contain a key enzyme for photosynthesis called Rubisco. These compartments have a mechanism for concentrating CO₂ 50 times more than other land plant chloroplast which leads to a much more efficient Rubisco and photosynthesis altogether. Introducing the genes involved in developing pyrenoids into crops could lead to greater efficiency and greater yield. Improving crops to sustain growing populations and providing for the underserved are significant matters that can be addressed by diving into hornwort characteristics. Genetic tools such as gene transformation and gene editing are vital for identifying the genes involved in pyrenoids, however, these tools have yet to be routinely implemented in hornworts. We are investigating the biolistic method of gene transfer by creating a protocol that is specific to hornwort tissue. The aim of this project is to optimize the biolistic gene transfer method in Anthoceros agrestis in order to consistently and efficiently perform genetic transformation experiments.

My Experience:

This summer I learned about what it is like to be a full-time researcher which is not something I could learn from my school. I learned new lab techniques that I can bring back to my lab at home, but more importantly, I learned how to set up an experiment and ask scientific questions. Throughout my interactions this summer I also learned skills dealing with interpersonal relationships with mentors, PIs, and other researchers in my lab. I know what to look for in a lab that I can work well with. The information I gained about grad school and the grad school application process I will take with me when figuring out my next steps.

“Investigation of a hornwort insertion line with rhizoids specific GFP expression”

Project Summary:

Hornworts share an ancestor with vascular plants around 500 million years ago and serves as the keystone for understanding the evolution of all vascular plants known today from their algal ancestors. Hornworts also have a symbiotic relationship with cyanobacteria and contain pyrenoid structures that sequester more carbon than chloroplasts of vascular plants. Doing more research on hornwort genomes will therefore aid in our understanding of these characteristics so that they might be implemented in crop plants to increase agricultural yield. There is not much coverage of hornworts in the literature, so preliminary investigation of these hornwort genomes is needed. This investigation looks at the rhizoids of the model species Anthoceros agrestis after it was bombarded with a plasmid containing the fluorescent gene GFP via a gene gun. Though the plasmid containing GFP was bombarded in all tissue, only the rhizoids of this specific strain fluoresced under UV light. To try and understand this, RT-PCR was performed on rhizoid tissue on the genes surrounding the insert to see if they are being expressed and acting as a promoter for GFP. This test showed the genes present and expressed in rhizoid tissue. The DNA of the strain was also sequenced using Nanopore and Readfish Selective Sequencing to gain longer reads and more depth on our target insertion site. The DNA sequence showed multiple inserts of the GFP gene as well as its promoters. A pairwise comparison of the syntenic relationship of the genes surrounding the GFP insert showed the genes belonging to a very large syntenic block conserved in all the genomes of hornworts. GO enrichment factors of this syntenic block were examined and showed an enrichment of DNA-transcription factors. This leads us to believe that the genes surrounding the GFP insert are epigenetically controlled (i.e., methylation or histone acetylation) which is consistent with these gene’s large synteny and enrichment factors.

Exploring the Metagenome of the Harmful Algal Bloom Community in Cayuga Lake

Harmful algal blooms (HABs) form green films that cover lakes and rivers during warmer months. Such blooms can last from a few short hours up to several months and come at a profound ecological and economic cost. Cyanobacteria are one of the contributors to such HABs. The films they form can block sunlight and deplete oxygen needed by plant and animal life. Additionally, cyanobacteria release neurotoxins that can damage mammalian nerve tissue, posing a serious danger to both humans and aquatic mammals. HABs are primarily triggered by a surplus of nutrients in the water, nitrogen and phosphate in particular. Runoff from excessive fertilizer use in agriculture combined with increasing temperatures due to global warming only increase the frequency and severity of these HABs. Despite this, we have limited knowledge of the microbial diversity of HABs. Modern sequencing technologies, however, can elucidate these community compositions. We aim to analyze the metagenome of the Cayuga Lake waters to draw associations between microbial compositions and future HABs. Metagenomic sequencing using a nanopore MinION DNA sequencer revealed that 41% of metagenomic reads were assigned to cyanobacteria. Based on ribosomal 16S sequences, the major cyanobacteria is likely to be Dolichospernum spp. In addition, 52% of the reads belong to proteobacteria orders including Rhizobiales (19%), Sphingomonadales (11%), Burkholderiales (4%), Rhodospirillales (3%), and Pelagibacterales (3%). This study is one of the few that characterizes the metagenome of HABs. These results will contribute to future research linking microbial composition with cyanobacteria blooms. Further studies are necessary to examine how these organism interact to form HABs and their ecological effects.

My Experience

I can happily say that I have gained so much from my summer spent at Boyce Thompson Institute. Under the guidance of my mentor, Dr. Fay-Wei Li, I was able to learn a variety of new lab techniques and procedures, all while being encouraged to become a better problem-solver and a more independent researcher. Through my project, I had the rewarding opportunity to see my efforts come full circle, taking part in every step from start to finish. Outside of my work, the resources I acquired and connections I built have been invaluable. It was a treat to share insight with my fellow interns and lab members, as well as gain practical wisdom and advice from the faculty and professors I met. Their energy and excitement for their scientific work was infectious and these people have only spurred my passion for research and plant biology. I feel confident in my ability to succeed in a scientific career and will never forget the amazing experience I had with the REU at BTI.

Exploring the bacterial endophyte communities of Lycopodiaceae

Plant-microbe interactions have been instrumental to plant ecology since the first plant terrestrialisation over 400 million years ago. Endophytes, defined as microbes living within healthy plant tissue, can increase host plant competitive fitness, confer resistance to biotic and abiotic stress, and promote growth by producing phytohormones or providing essential limiting nutrients. Lycophytes are the earliest diverging extant lineage of vascular plants and evolved roots independently from the other vascular plants (ferns and seed plants); roots are an important entry point for endophytic microbes. The lycophyte microbiome has been scarcely characterized, leaving a significant knowledge gap both in the evolution of plant-microbe interactions and in the biochemistry and ecology of lycophytes. Many lycophytes are under conservation status and some species (Huperzia spp.) produce Huperzine A, a compound with potential application as an Alzheimer’s medication. Understanding the microbiome would lend insight to explanations of lycophyte distribution patterns and biochemical properties, aiding conservation and medical applications.
We collected samples of five species in the family Lycopodiaceae from a single population at Shindagin Hollow State Forest near Ithaca, New York: Lycopodium clavatum, Dendrolycopodium dendroideum, Spinulum annotinum, Huperzia lucidula, and Disphasiastrum digitatum. We surface sterilized plant segments of different tissue types, plated them on LB agar, and isolated single bacterial colonies that grew. We then amplified and sequenced the 16S region (primers: 27F and 1492R) of each bacterial isolate to identify bacterial taxa. Overall, the most frequently occurring bacterial genus was Bacillus followed by Paenibacillus. The greatest isolate count and bacterial diversity was found in H. lucidula. Subterranean tissue samples produced over twice the number of isolates as aerial tissue samples. With taxa abundance data, we utilized biodiversity and similarity indices to determine host and tissue specificity. Based on previous research, the bacterial endophytes identified include known plant-growth promoting rhizobacteria, phytopathogens, root-associating and endophytic diazotrophs (nitrogen-fixers), and bacteria that confer stress tolerance to their hosts. Future research could look into the specific roles of these bacteria in lycophyte ecology or perform more extensive analyses to identify phylogenetic patterns in the evolution of lycophyte-microbe associations.

My Experience

These past two months at BTI have been some of the shortest months of my life. I think it has felt this way because my time has been filled with doing interesting research, making new friends, learning new things through various BTI-sponsored activities, and exploring a part of the country I have never been before. Life has been a beautiful balance between taking strides to achieve life goals and having a good time along the way. As my first research position, working in the Fay-Wei Li lab taught me what science in action is like: the trial and error, the repetition, and the feeling of satisfaction when you get results that mean something. I experienced just how much researchers know about their field and the ways that they are actively expanding it. Before this internship, I was on the fence about whether or not I wanted to attend graduate school. Now, I am confident in my desire to continue researching the things that inspire fascination in me as well as have the potential to help people and our environment. Being at BTI has made my next steps in life clear to me: I am going to take a gap year after graduation to gain more research experience before attending graduate school in a field of the environmental sciences.

“Nitrogen fixing symbioses in bryophytes show host preferences between cyanobionts”

Project Summary:

Nitrogen is important for all plants but it is often a limiting nutrient because plants cannot directly metabolize the nitrogen gas that composes 78% of the earth’s atmosphere. (Goyal et al. 2005). In order to obtain nitrogen when not enough is available in the soil, certain plant species have developed symbiotic relationships with bacteria that fix atmospheric nitrogen, converting it into compounds the plants can process. The most ancient origin of bacteria-plant nitrogen fixing symbiosis is that of the hornwort-cyanobacteria symbiosis.  Hornworts are one of the three lineages of bryophytes—hornworts, liverworts, and mosses—all characterized by their gametophyte dominated life cycle, lack of vasculature, and preference for moist cool habitats. Within the bryophytes, all hornworts and two liverwort species can form symbiosis with cyanobacteria. The cyanobacteria, mostly in the genus Nostoc, live in mucus cavities in the plants. Studying this symbiosis will help elucidate the evolution of nitrogen fixing symbioses in plants. Our study investigates the variation in symbiosis between different pairings of cyanobacterial strains and bryophyte hosts grown in microcosms. In this experiment we inoculated sterile plants of the four bryophyte species Anthoceros punctatus, Anthoceros agrestis, Blasia pusilla, and Dendroceros sp., with two cyanobacteria of the Nostoc genus: Nostoc punctiforme and an unidentified Nostoc species. We measured the growth rates of the plants and how many colonies the cyanobacteria formed in the hosts. Although the cyanobacterial strains did not affect host growth, colonization rates consistently differed between the two Nostoc strains, demonstrating host preferences between symbionts.

My Experience:

A transformative experience is how I would dub this internship. The best part of it is was not what I learned about PCR mixes, bench work, Acetylene Reduction Assays, primer design, non-vascular plant taxonomy, or any number of new lab techniques, but what my fellow interns and co-workers taught me about what it means to carry out science and gain knowledge from it. The passion that I saw science fueling in the eyes of people around me allowed me to realize how much it enriches the lives and spirits of those who choose research as their purpose. Listening to the practical and scientific wisdom of my peers, mentors, and co-workers taught me many things I did not know existed. This experience has made me more passionate to learn in academic research settings and continue doing science!