Blog Updates

Art by Beatrix Potter

Mycological madness

November 28th, 2022

Although I’m sure readers everywhere have thoroughly enjoyed the action shots of Sarah, Marvin, and I, skillfully pipetting, like wizards casting spells with their wands - pictures have limits to the amount of entertainment and information they can provide. So, as we wait for the stress and NSC results to be packaged into something interpretable for the blog, I've made the executive decision to cover a topic seemingly unrelated to waterlogging - the history of mycology! This deep dig will explore the foundational discoveries and scientists within the fungal field. We're also going to investigate how the natural world communicates with artists, impassioning creatives across the globe to exceed the limitations of physical images by casting stories and connecting diverse concepts in the minds of their viewers.

The young science of an ancient kingdom

Close your eyes and try to imagine yourself in the ancient city of Athens, sitting next to one of the only Greek writers whose works survived the fall of the Greek empire and the natural decay of artifacts that would take place over the next 2,500 years. The man, named Epicharmus, leans over to you and says dryly, "You are like mushrooms: you will dry me up and choke me to death." Epicharmus was quite charming after all, building a career as a dramatist takes a certain kind of comedic prowess. Society's constant pull towards satire and humor coincides with our interest in the natural world. As Greek philosophers were philosophizing about humanity, meaning, and morals, they pointed towards objects in nature to convey their thoughts. These pontifications provided us with our oldest written evidence describing mushroom species and uses. It seems as though the sapien interest in fungi, just like Epicharmus's diary entries, blog posts, or whatever medium he preferred, has stood the test of time.

Open your eyes and fast-forward around 3 millennium, you're now sitting next to a cheeky blogger who gets a little too much joy out of subjecting her readers to time-travel and the history of all things fungi related. If you can remember back to my earlier posts on fungi, I have covered the following: when and how mushrooms grow, different types of mycorrhiza (arbuscular mycorrhiza: AM, and ectomycorrhiza: EM), edible mushrooms, and the history of mushroom cultivation. Today I'll dig into a topic that is relatively new to the scene when considering the fact that mushrooms have been growing on Earth's terrestrial surfaces for over a billion years and humans have been cultivating them for thousands. We are talking about the field of mycology, folks. From the first dude to categorize fungi apart from plants in the 18th century - to the exciting and novel fungal mapping being done today by researchers such as Giuliana Furci and Toby Kiers, the fungal thread will be followed!

Let us begin with Pier Micheli and Elias Fries. With the publishing of his book Nova Plantarum in 1729, Micheli became the first botanist to group grasses, mosses, and fungi using systematics. Because of his prescient classification skills, Micheli was donned the father of mycology. The next breakthrough in the field wouldn't come until a century later when Elias Fries released Systema Mycologicum to a highly anticipatory audience of fungi fanatics in 1821. I may be playing-up the enthusiasm for mushroom content at the time, as the general public wasn't too interested in mycology until later practical applications were discovered such as Eduard Buchner's extraction of zymase in 1897 or Alexander Flemming's invention of the first antibiotic, Penicillin, in 1928. Nevertheless, Fries was referred to as the Linnaeus of mycology (what's up with these guys and all their nick-names?) because of his major contributions naming and identifying many species. He used characteristics such as spore color and gill type to group mushrooms, ID traits that you can find foragers and mycophiles still using in present-day.

On the topic of innovations we still use today - Lewis Shweinitz, the Tulasne brothers, and Anton de Bary, were other influential pioneers in the field of mycology who would go on to make lasting impacts in how we identify, name, and biologically understand fungi. Shweinitz listed 1,300+ species in his 1822 book Synopsis Fungorum Carolinae Superioris, which served as the first thorough guide to fungal species in North America. The Tulasne brothers revolutionized our scientific understanding of fungal polymorphisms and reproduction cycles throughout the mid-1800's. Anton de Bary followed this up with his innovative work with pathogenic fungi and heteroecism. Being a man of many talents, he was given many names. These include the founding father of plant pathology (history really has a thing for dads), and the founder of modern mycology. I can't overstate the importance of Bary's contributions to science, as he truly dedicated his life to his studies and worked to apply his research to contemporary social issues such as the potato blight causing severe crop loss and economic devastation in 1860's Germany. Bary also provided novel insights on fungal sexuality, lichens, and experimental methods that continue to be implemented by botanists and bacteriologists currently.

The latter half of the 19th century saw significant contributions from Pier Saccardo and Albert Frank. I know what you're all thinking: "Are white guys the only demographic of scientist interested in fungi?" We should all take a moment to consider the lack of diversity in the field of mycology up to this point, not only between sexes, but also between ethnic and social classes. The inclusivity increases a little once we get into the 20th century, but I'll also address how we can all work towards recognizing the accomplishments of scientists from marginalized groups. Back to Saccardo, who in 1872 published Mycologiae Venetae Specimen, a comprehensive guide that described over 1200 species of fungi. His descriptions of fungi in the group Deuteromycota and in the class Pyrenomycetes were additional contributions to the fungal classification system. Shortly thereafter in 1885, Albert Frank coined the term mycorrhiza. Thanks to Frank and his mycorrhizal obsessed contemporaries, the fungal kingdom's ecological importance began to garner serious attention, and the wide-spread association of EM fungi and their tree hosts became the focal point for many forest and plant researchers. Frank's original hypotheses involving water and nutrient exchange between fungi and plants would be expanded upon in the 1900's, and continue to hold true today.

Lexikon Blogion

Systematics: Just a reminder for those of you who have forgotten our previous run-ins with Mr. Linnaeus, systematics is the study of the diversity and relationships between past/living organisms. This field uses the naming and identification systems of taxonomy and phylogenetic trees to view relationships, traits, time-scales, and so on. Fun fact about our pal Pier Micheli, he coined the genus names 'polyporus' and 'tuber,' which we still use today.

Zymase: the enzyme responsible for the fermentation process in yeast. What exactly is yeast, you ask? Yeast is a single-celled fungi that humans have found particularly useful, especially in the production of alcohols and breads.

Penicillin: an antimicrobial compound originally obtained from the green mold species Penicillium notatum. Estimated to have saved over 200 million lives since its first use as a medicine in 1942, Penicillin helped sufferers of scarlet fever, pneumonia, tuberculosis, syphilis, and more.

Polymorphism:Poly meaning 'many,' and morph meaning 'shape.' Polymorphism refers to organisms whose DNA codes for multiple shapes, depending on the environment. In the case of fungi, they can exist as unicellular species that later form multicellular filamentous networks. A fungi's hyphal structures can also change shape in response to host and environmental factors.

Heteroecism: refers to the different developmental stages a parasitic organism undergoes while completing it's life-cyle across two hosts. Most species of rust fungi spend their adult life on the primary host, while the alternate life-stage is spent on secondary host of another species. Parasites requiring only one host are called autoecious.

Lichen: a complex and amazing combination of fungal filaments paired with green algae (Chlorophyta) or cyanobacteria (Cyanophyta). The outer body of the lichen is formed by the fungi, for which each species is named. Algae and/or cyanobacteria set up shop underneath the protective fungal body (called the 'thallus'). According to taxonomists, up to 30% of fungi species can become lichens. Fun fact about our friend Anton de Bary: he coined the word "symbiosis" in 1879 when describing the mutually beneficial relationship fungi form with bacteria and plants.

Deuteromycota: a group of fungi, also called 'imperfect fungi,' that are unable to be classified using common morphological characteristics because their sexual reproductive stages are unknown.

Pyrenomycetes: class of fungi within the division of Ascomycota. These fungi have small, flask-shaped fruiting bodies and mainly consist of decomposers and plant/animal parasites.

At the turn of the century, Elsie Wakefield and Johanna Westerdijk not only made major contributions to the field of mycology, but served as excellent role-models for how to contribute to the scientific community beyond the influence of research. Through my online sleuthing, I found no mention of anyone being dubbed the mother of mycology (as we know, there are so many fathers!). So, by the power vested in me, based on no qualifications or reasoning that would hold up in a court of law, I hereby declare both women official mothers of mycology! Now that the ceremonial proceedings are finished, we can return to 1910 when Wakefield began her position at Kew, the Royal Botanic Gardens in London. Wakefield would go on to publish over 100 papers on fungal reproduction and phytopathology, in addition to her beautiful illustrations of fungi that can be found preserved at Kew and in her two field guides on British fungi. She would go on to serve as the Head of Mycology at Kew for 40 years and was elected as President of the British Mycological Society in 1929. In 1917, Johanna Westerdijk became the first female professor in the Netherlands. Just like Anton de Bary before her, Westerdijk's work focused on the immediate social, economic, and environmental, issues of her time. Her most notable contributions include her research on potato diseases, dutch elm disease, and the fungal taxonomy system she developed. Westerdijk also emphasized the importance of mentorship and inclusion in science by running a lab full of ladies. She mentored 56 PhD candidates, almost half of which were women. Check out this website to learn more about Johanna, and look into Annie Smith and Lilian Hawker if you're searching for more tea on the British Mycological Society and influential women in science during the 1900's.

While Wakefield and Westerdijk focused on fungi induced plant diseases, the mycorrhizal boom ignited by Albert Frank in the previous century was stoked by Erik Bjorkman, John Harley, and Barbara Mosse. In 1942, Bjorkman developed the carbohydrate theory of mycorrhiza symbiosis. Bjorkman's experiments showed higher frequency in mycorrhizal association with conifers when carbohydrate concentrations were increased in fine roots. In 1959, Harley revolutionized our understanding of mycorrhiza physiology when publishing his book The Biology of Mycorrhiza. Phosphate and nutrient exchange in these symbiotic systems were then pushed to the fore-front. Harley's students would go on to classify the dominant mycorrhizal types associated with different plant communities. This is an ongoing focus of research groups that I will return to at the end of our walk down mycelial lane. Meanwhile, Barbara Mosse wouldn't allow herbs and flowering plants to be left in the dust dirt. In 1962, she established a protocol for cultivation of mycorrhiza in laboratory settings that is still followed by fungi farmers and mycological researchers presently. In collaboration with Roger Koide, Mosse would later publish “A history of research on arbuscular mycorrhizas'' that thoroughly synthesized that impacts of AM fungi on the plant kingdom.

The omics revolution during the 1990's allowed researchers to identify fungal symbionts via RNA analysis, and duos such as Maria Harrison and Marianne van Buuren to describe the molecular basis of fungal nutrient uptake processes. Using RNA ribosomal genes to ID fungi is a much more efficient approach because of the microscopic nature of most mycorrhiza species and the difficulty in distinguishing between their morphological characteristics. In 1995, Harrison and van Buuren discovered fungi phosphate uptake pathways by comparing the DNA and subsequently coded proteins of different fungi species. They were able to confirm the function of a protein found in Glomus versiforme (an AM fungi) by observing the impacts of the complementary gene in yeast cells, which codes for a trans-membrane phosphate transporter. I was unable to access their paper, but I provided an image depicting mycorrhizal phosphate pathways from a more recent publication.

Omics-based research chugged along during the early 2000's, aiding scientists in the discovery of root signaling molecules that facilitate mycorrhizal colonization, whole-genome sequencing of fungi species, and relocating fungi species on phylogenetic trees to more accurately represent their evolutionary relationships. In 2005, Akiyama and colleagues isolated compounds from root exudates, previously known as branching factors, that induced morphological changes in symbiotic fungi proceeding root colonization. I will return to these signaling molecules, which Akiyama identified as strigolactones, in the next blog update. In 2008, Martin et al. published findings from a genome sequencing project that revealed a number of surprising proteins synthesized by the EM fungi Laccaria bicolor. The fungus was found to release various small secreted proteins (SSPs) with unknown functions and enzymes that were able to degrade non-plant cell wall polysaccharides. The authors predicted that the SSPs help to facilitate mycorrhiza colonization of plant roots, and the enzymes allow for the fungus to live freely in the soil, feeding off of soil microbes when carbohydrates from a host plant aren't on the menu. Genome sequencing such as this has further informed the renaming and reclassifying of fungi within more accurate groups based on genetic relatedness.

In search of interesting mycological research happening at this very moment, I found two articles covering both exciting science and scientists. First up we have self-taught mycologist William Padilla-Brown and his work on Cordyceps cultivation. Until Padilla-Brown's independent research efforts, Cordyceps species, used for the medicinal benefits, were only grown in large-scale operations in China and other regions of Asia. Through a process of finding specimens in the wild, collecting their spores, and breeding them in sterile conditions, William learned how to cultivate the fungus on substrate other than their preferred insect hosts. He then published a guide on Cordyceps cultivation and continues to lead education courses on the subject. The next article follows mycologists Giuliana Furci (founder of the Chilean nonprofit Fungi Foundation) and Toby Kiers (VU Amsterdam) as they work with the Society for the Protection of Underground Networks (SPUN) to map the global distribution of mycorrhizal fungi. SPUN's big aspirations will surely be a difficult feat, but it is vital work, as many countries have yet to include fungal species on endangered species lists, or even consider them as organisms that should be protected. Both William Padilla-Brown and Giuliana Furci also serve as great examples of non-traditional routes to careers in research, both being self-taught and successful without the conventional academic training.

Lexikon Blogion

Phytopathology: a fancy word for plant pathology, which is the study of plant diseases caused by pathogens or environmental factors.

Omics: technologies that all scientists to study biological systems from a holistic view using high-throughput sequencing of biological molecules. Leroy Hood and colleagues first developed DNA sequencing and synthesizing in the early 1990's. Soon thereafter, scientists could look at whole genomes using genomics, metabolites using metabolomics, RNA molecules using transcriptomics, and proteins using proteomics.

Strigolactones: signaling compounds synthesized by plants that serve two main functions: hormonal control of plant development and initiation of mycorrhizal symbiosis by functioning as a signaling molecule after exudation.

Cordyceps: a parasitic genus of fungi within the Ascomycota division. Found globally but most prevalent in humid temperate and tropical jungles, this diverse genus contains around 600 species. Their hosts include insects, arthropods, and a few fellow fungi.

An image from the fungi phosphate uptake paper I linked. This is showing intraradical colonization of AM fungi. Senescent colonization is referring to collapsed arbuscules which usually appear 1-2 days after initial colonization. The function of these structures are unknown.

Cordyceps, otherwise known as the cheetos of the forest. Image source.

This image shows all the interactions, both beneficial and pathogenic, that strigalones facilitate. Image source.

Mushroom Art

What inspires art and science? What is the deep, humanistic urge, to understand and explore the world both around and beyond us? It is through the intense concentration on the minute details, the characteristics that seem disposable and nonessential, that imaginative minds cling onto and expand. Through this expansion comes an understanding of the whole, and understanding breeds connection - the inspiration behind most human endeavors.

Up first we have Beatrix Potter's drawings of Flammulina velutipes, Himeola auricula, and Hygrophorus puniceus. Potter, famous children's book author of The Tale of Peter Rabbit and other popular works, developed an interest for mushrooms in her early twenties. Images are from the Armitt Museum and Library.

Next we have drawings of the previously mentioned Elsie Wakefield. The individual mushrooms illustrated first include Amanita muscaria, Morchella spp., and Calvatia gigantea. All sketches are kept at the Kew Royal Botanic Gardens.

These are all embroidered pieces from artist Amanda Cobbett. You can find her work here. Cobbett's work is inspired from observations of the understory and forest floor.

Last but most certainly not least, we have a Root Lab original from our very own in-house artist, Sarah Romy. Historians have scoured through Romy's journals looking for the source of her inspiration. Apparently this particular work came to her in a vivid dream.

Art by Henning Wagenbreth

The lives of lab rats

November 11th, 2022

We set up shop in the Root Lab this past fortnight and powered through some intense pipetting sessions for the stress and NSC analyses of our waterlogging samples. We'll still be running NSC tests late into next week, so I don't have any results to share for the time being. What I do have is pictures, and I will post them in abundance for any of you curious readers who dream of one day performing thumb-straining micropipetting work until your phalange tendons snap. After being pulled through a virtual tour of all our recent lab work, what better way to exit the ride than a one-sided discussion where I elucidate my thoughts on Dr. Victor Frankenstein and ethics in science.

Benchwork

What is benchwork? I was unaware of the term myself prior to working at Morton. Most of the labs during my undergrad studies were relocated to virtual spaces in response to COVID-19, so I guess I was doing laptopwork. For my American readers out there, the word "bench" is synonymous with "table." Before you ask, yes, it was important for me to directly address my American readers because 1) I have a large international audience that checks in regularly, thank you very much, and 2) from my five minutes of googling, the term only seems to cause confusion for myself and a few of my fellow US citizens. Australians and the guy on the Great British Bake Off use the word all the time (according to reddit). I digress, anyway, benchwork is carried out in a laboratory setting using lab benches, sinks, fume/tissue culture hoods, microscopes, and so on. To get really technical, everything I listed above would be considered "wet" lab research, whereas computational work and anything else that can be done in an office setting is defined as "dry" lab research.

The last couple of weeks we have been wearing out the benches in the Root Lab. I know it's unnecessary to summarize what we've been up to over the past month, as you have all been reading along devotedly, I'm sure. From the grinding of root and stem samples, to the weighing of samples and subsequent stress and NSC tests we've been running, the waterlogging project has provided us with a substantial amount of wet lab work. I suppose it's only fair that we endure the same saturated conditions as our poor trees. Below are some pictures and descriptions detailing the stress and NSC analyses.

Above you can see the steps that went into the peroxidase test. A protein extraction buffer was made and kept on a magnetic stirrer to keep the buffer from separating. At multiple points during the peroxidase protocol, we also used a centrifuge to separate the liquid (containing the extracted proteins) and the tissue in the microcentrifuge tubes. Samples were also sonicated in an ice bath. The last images show the 96-well plates with the added guaiacol reagent, the octo-pipette, and the spectrophotometer plate-reader that measures the absorbance levels of the samples.

These are samples pipetted into the 96-well plates for the TBARS test. If you can recall, this test is used for quantifying the oxidative damage in the fine roots. The darker yellow reactions signify higher MDA (byproduct of lipid oxidative damage) concentrations.

Imagine an endless montage of pipetting playing over inspirational music. What you see here are only half of our NSC samples (the fine roots). We will be running the coarse root and stem NSC analysis next week.

Mary Shelley and ethical science

Near the end of summer in 1797 Somers Town, London, Mary Wollstonecraft Shelley, whose relentless imagination and prescient thoughts on life and the human condition would continue to influence readers centuries after her own departure, was born. The womb from which she came belonged to that of the revolutionary writer and early feminist, Mary Shelley. Her father, prominent philosopher William Godwin, was Mary's sole parental figure, as her mother passed just 11 days after her birth. Constantly surrounded by her father's intellectual guests, she was absorbed in the philosophical, religious, and scientific views and literature of the era. At the time, turmoil seemed to be a running global theme, as the American, French, and Industrial Revolutions were all beginning or ending their revolving motions. The reverberations of the Enlightenment, along with innovations in science, were shaping the world. The amalgamation of these happenings, paired with the wonder and curiosity within Shelley herself, would be combined internally to synthesize her most famous novel: Frankenstein; or, The Modern Prometheus.

This 1831 painting by Samuel John Stump is titled Unknown woman, formerly known as Mary Shelley. How mysterious. Image source.

A short and somewhat satirical summary of Shelley's story: Dr. Victor Frankenstein was raised in Geneva, Switzerland by a nurturing and supportive family that seemed to love him too much. From a young age, he had an intense thirst for knowledge and the origins of life. He went to Germany and studied chemistry after his mom passed, quickly falling into the obsession of creating life himself in the form of a human-like creature. After years of isolation and hanging out in grave yards, sleuthing for spare parts like a fanatic antique collector, he reaches his goal and brings the unnamed thing to life via sutures and electricity. In an impressive act lacking any hint of forethought, much like all of his actions up to this point, Victor also decides to make the creature super huge and tall and strong. What could go wrong?

Spoiler: a lot could go wrong! Vic unsurprisingly runs away from his creation in a fit of terrified regret, and the creature traverses into the countryside like a neglected orphan, only to be met with prejudice and fear from everyone he encounters along the way. This cultivates a deep and bitter hatred in the creature towards mankind. Victor's creation then tracks him down and demands that the inventive scientist build him a girlfriend. When Vic refuses, a series of tragic events unveil and the creature kills everyone whom Victor loves. The splendid and joyous fantasy ends with Victor warning another scientist not to repeat his mistakes in the quest for knowledge, and the creature crying over Victor's corpse before running away into the Arctic wastelands.

Frankenstein was first published anonymously in 1818. Image source.

Frankenstein is, at its core, a novel exploring the boundaries of human knowledge and creation and the consequences that arise from wielding those two instruments. Personally, I also think Shelley is pointing to the beauty and purpose of life that is beyond what we are able to observe or discover through science and analytical thought, but we can consider her romanticist leanings later. Frankenstein has been reinterpreted by a multitude of playwrights and directors over the past two centuries, to the point where Frankenstein's creation has become a meme far off from the original character Shelley stitched together. The screws and green skin were later additions that obstructed the author's original intent to illustrate the creature as sophisticated and sensitive, yet just beyond the reach of human compassion and connection. Dr. Frankenstein brought life into an uninviting world, and in that act of creation, far surpassed his own abilities to control the being he shaped with his own hands. We know the turbulent historical context in which Shelley penned this literary staple, but how did her book fit in amongst its contemporaries?

Shelley's 1818 novel is considered by many literary critics to be the first true work of science fiction, as the narrative of the story is supported by an act of science gone awry, rather than magic or godly influence. Previous plays, poems, and novels had explored the dark side of human will and hubris. For example, Shakespeare penned dozens of plays on the topics beginning in the 1590's and into the early 1600's. John Milton's 1667 Paradise Lost was an epic poem that greatly influenced Shelley's work. However, Frankenstein was such a paradigm shifting piece of literature because it used science as a vehicle to explore such maladies of the human condition, prompting readers everywhere to consider mankind's potential to cause irreversible damage through experimentation and invention. Shelley's romanticism influences presented a duality between the purely analytical mind born out of the Scientific Revolution and the Enlightenment, with the intrinsic beauty and potential of both the individual's imagination and nature. This untethered curiosity is what leads to such influential works of fiction, and fiction allows us to explore future scenarios that are rarely predicted by those who are firmly planted in the present. In every year since, the book has resonated more and more as innovations in technology and scientific understanding spur discussions around the ethical uses of such innovations.

What is fair and sustainable? What promotes compassion and demotes suffering? These questions should be asked when considering applications of science and technology such as the atomic bomb, artificial intelligence, genetic engineering of humans and other animals, genetically modified organisms (GMOs) in our foodstuffs, and industrial agriculture methods, just to name a few. These topics are far reaching and have global implications. They are all interesting and worthy of consideration and discussion. However, I am biased, and whether it is due to my innate interests or the current ecological research I do now, I find the impacts of science and tech on the environment, our agricultural systems, and food production, to be most compelling. In what ways have we as a species, like Dr. Frankenstein, acted without forethought?

A provocative and informative book that Shelley's tale of human hubris reminded me of was Charles C. Mann's The Wizard and the Prophet. I will spare you all from another unwarranted book synopsis, but I will point to Mann's work here because it provides a detailed historical account of western science and society's relationship with the environment. The Wizard and the Prophet is a fantastically written dual biography on two prominent scientists of the 20th century, Norman Borlaug and William Vogt. Mann argues that both men laid the foundation for two opposing philosophies: Borlaug, a "wizard," believed that harnessing the power and knowledge available through science and technology would help shape our world for the better, whereas Vogt, a "prophet," preached of the importance of using science to reveal our biological and environmental limits and responsibilities. Mann details innovations such as The Green Revolution, a turning point in global agriculture production largely influenced by Borlaug himself in the late 1960's, to explore the opposing views on scientific progress and its impacts.

Borlaug, through years of admirable dedication and impressive work on selective breeding programs that produced high-yield and disease resistant wheat varieties in Mexico, would apply his life's work to save over a billion starving people worldwide. For any readers who are interested in crop science, plant breeding, or plant pathology, I recommend checking out his story. Using Borlaug's seeds, paired with chemical fertilizers, pesticides, and amended irrigation regiments, countries such as Mexico and India were able to become independent producers of cereal grains. In the East, China and the Philippines would adopt the same methods with high-yield and disease resistant rice varieties. Borlaug and Co. would go on to ship their seeds and cultivation methods to Columbia, Ecuador, Chile, and Brazil. For all of this work, Borlaug was awarded the Nobel Peace Prize in 1970.

Criticism of Borlaug's work wouldn't come until decades later when environmentalists and social critics would begin questioning the implications of the Green Revolution. The intensive production of crops that Borlaug and colleagues' varieties and cultivation methods required high-inputs of water and agrochemicals, leading to epidemic environmental degradation. There were also social ramifications that led many small-scale farmers to shoulder increasing amounts of debt, if not retiring from the career altogether. In The Wizard and the Prophet, Mann goes on to question who is responsible for overseeing the applications, as well as the intended and unintended impacts of scientific and technological innovations. Is it the researchers themselves? Those funding the research? The private and governmental bodies that end up using the knowledge and technology gained from such studies? The most logical and prudent option may be all of the above.

There are laws, policies, and peer review systems in place to ensure ethics in science are upheld during the scientific process and throughout the subsequent application(s) of the research. For Dr. Frankenstein, these systems were not set in place. The peer-review process was established in Europe in 1731 by the Royal Society of Edinburgh, which didn't provide much direction for a young Dr. Frankenstein, who didn't seem too interested in publishing papers. In terms of actual legislation, the US didn't require Institutional Review Boards at research centers until the passing of The National Research Act in 1974, and Europe's 1947 Nuremberg Code wasn't too far ahead. As consumers of science and technology, we can do things (e.g. volunteer, forward an interesting science article to a friend, vote for government officials who are science supporters) to help support research that we believe leads to the betterment of society. As scientists, it is our responsibility to refrain from becoming engrossed in the quest for knowledge and discovery, and to consider the consequences of both our own work and that of our peers.

Trick or trees

October 31st, 2022

The spookiness is at an all time high and a seasonal force disrupted the Root Lab's game plan this week. After a few minor explosions and other mishaps, we finally got our stress tests running on Thursday. The late start pushed our NSC analysis back a week, but don't you worry, I'm fulfilling my promise and explaining the process in full, no more cliff-hangers! Keep reading to find out more about how we measure our woody friends' energy reserves and a special Halloween treat exploring the complex history of "witchcraft" and spell-binding plant potions.

The ABC's of NSCs

If you can remember all the way back to the blog update on August 5th when I first explained how plants store and use nonstructural carbohydrates (NSCs), then I applaud you for your great recall and dedicated readership. If not, then please begin reviewing this material every night before you go to bed. Someday a random stranger will surely ask you about NSCs, and your preparedness for such a question might determine the fate of the universe. Okay, back to the carbohydrates.

NSCs play a central role in many plant metabolic processes. They are the main product of photosynthesis, like McDonald's French fries or Levi's jeans. They provide the energy for plant biosynthesis and are therefore involved in most all physiological processes. Scientists have just begun to explore the influence of NSCs on plant response to environmental change, specifically regarding drought. They also happen to be a reoccurring guest on this blog, so let's get into their molecular structure and how we can measure them in plant tissue.

Plants biosynthesize numerous compounds that we classify as NSCs. All are molecularly light weight mono- and oligosaccharides (simple sugars), polysaccharides (starch), and/or fructans. Below are some examples of carbohydrate molecules (Image source). There are many methods to measure NSCs, some are better at extracting and quantifying specific compounds, so deciding on which protocols to use depends on which compounds you want to target in your analysis.

The three main methods of NSC analysis are ion chromatography (IC), enzymatic conversion, and oxidation via concentrated acids. IC separates and quantifies a wide range of mono- and oligosaccharides, as well as any present sugar alcohols in mixtures (sugar alcohols are another NSC compound synthesized by some plant taxa). Mass spectrometers can then be used to identify the separated compounds. Enzymatic methods quantify the simple sugars (sucrose, fructose, and glucose) in mixtures. They do this by converting the sugars into molecules that can be monitored at a certain wavelength using a Ultraviolet-Visible spectrometer (UV-Vis spectrometer). We chose to go with the acid method for our study because acids oxidize all water-soluble sugars and storage NSCs. The concentration of the oxidized products are measured by adding color-producing reagents and running the samples through a spectrophotometer, just like we did with our stress tests last week. We will be using phenol-sulfuric acid for the reagent because that method has shown the best results in previous studies. Speaking of results, what are we expecting to see in our trees?

Eons ago, in a blog far-far-away, I first introduced the different 'root strategies' that some of our trees may employ to tolerate the stress of waterlogging. Whatever story our NSC analysis reveals will surely be an interesting one, but for now it remains somewhat of a mystery. We are particularly interested in observing how the stem and coarse root NSC pools shift after the recovery period for both magnolia species. Remember, we harvested stem and root tissue on the day we took the treated trees out of the kiddie-pools, and then again after a three-week recovery period. If rapid die-back of the root system occurs during sustained waterlogging, followed by a new flush of root growth once the environment is drained of water and reoxygenated, then the mechanisms of this strategy may be partially explained through our NSC results. Higher levels of NSCs in the stems and coarse roots during the earlier harvest date, and lower NSCs in the latter would provide evidence supporting this strategy of carbohydrate allocation.

Lexikon Blogion

Biosynthesis: simply put, biosynthesis includes all the processes in an organism's body that turns simple structures or molecules into more complex ones. The conveyer belts driving this synthesis are the metabolic pathways, and the factory workers are enzymes. Products can be modified, converted into completely different molecules, or joined to synthesize macromolecules.

Physiological processes: linked to biosynthesis, physiology involves all the inner-workings of an organism's cells, tissues, organs, and organ systems, and how they function to sustain life. Yes, plants have organs too (leaves, stems, roots, and reproductive parts), but they may be more viewer friendly than our own. Displays of delicately curated human organ bouquets on your dining room table typically leads to confused and worried house guests.

Monosaccharides: saccharides are the 'building units' that make up carbohydrates. They contain carbon, hydrogen, and oxygen atoms. Monosaccharides are the simplest of these saccharides, meaning they cannot be broken down, or hydrolyzed, to any simpler sugar. Examples include glucose, fructose, and galactose.

Oligosaccharides: carbohydrate chains containing three to ten simple sugars. Examples include sucrose, lactose, and maltose. The Greek words oligos and oligoi mean "small" and "few."

Polysaccharides: loooooong carbohydrate chains, the most abundant form of carbohydrate found in nature and food. These chains can be linear or branched and are linked together by glycosidic bonds. Polysaccharides can serve both energy and structural purposes. For plants and humans, starch is an important polysaccharide energy source. Glycogen is another you may be familiar with, as it is the main source of fuel for your brain and muscles (sorry plants, you miss out on this one). Structural polysaccharides include cellulose and chitin.

Fructans: long chains of fructose molecules, that (according to wikipedia) are produced in over 12% of angiosperms. Not every species of plant or tree synthesizes fructans as an NSC compound.

Ion chromatography (IC): a form of liquid chromatography that separates the molecules in mixtures based on their charge. An ion, or charged molecule, can be positively charged (cation), or negatively charged (anion). The resin, which is the solid matrix with attached ions to filter your sample, can be either positive or negative. Your sample's ions will attach to to the oppositely charged ions in the resin, leaving you with the remaining cations/anions to quantify.

Mass spectrometers: used to identify unknown compounds, quantify known compounds, and determine chemical properties of molecules. Mass specs do this by first converting molecules to gas-phase ions and then measuring the mass-to-charge ratio of those ions.

UV-Vis spectrometer: like the above, UV-Vis is a type of organic spectrometry that uses a machine called a spectrometer to beam a form of energy at molecules and then measures the response of said molecules. Mass specs use high energy electrons, while UV-Vis specs beam ultraviolet or visible light at whatever molecules you're analyzing.

Humanity's missteps and plants' magistery

I unknowingly bit off more than I could chew when selecting witches and botanical elixirs for a holiday themed post. As the costumed kiddos were running around my neighborhood, having also just bitten off more than they could reasonably masticate in the form of caramel filled chocolate bars instead of preliminary research, I sat on my porch reading horrifying details about witch-hunts for nearly six hours. I'm not sure what I was expecting - a couple of articles covering the Salem witch trials and a listicle of witchy herbal remedies might have been a more digestible read, but that's not what I found. Where does the myth of the broomstick wielding magician stem from?

Accounts of magic, sorcery, and witchcraft predate the witch trials by many centuries. Homer's Odyssey, published by Harper Collins in 800 BC, describes the character Circe as a witch. Archeological remains of Greek curse tablets have been found near fountains and grave sites. These spells were often used in sporting events and legal battles. Next time you see a discus thrower hauling around a huge stone tablet at the Olympics, you'll know what it's for. The familiar image of the old, malevolent witch, popularized by folk tales and Disney, wasn't first described until centuries later in the cold and desolate Alps.

The Celtic peoples and other medieval inhabitants of the largest and most extensive mountain range covering south-central Europe are said to have started the legacy of Bertha (also known as Perchta or Befuna). Bertha was depicted as an old, haggardly woman, who was cold in demeanor and possessed magical powers. She rewarded those who did good, and punished those who didn't fulfill their responsibilities. Her focus seemed to be on children and servants, particularly regarding their household and wool spinning duties. I don't want to be presumptuous here, but it kinda sounds like Bertha was a behavioral tool invented by distressed Celtic parents. Now that the origins of the witch have been explained, allow me to attempt to cram the horror of the witchcraze (15th-18th centuries) into the following paragraphs so that we can swiftly shift the topic to magical plants.

A 1550 painting by Pellegrino Tibaldi capturing a scene from Homer's 'Odyssey.' Circe seems to be turning men into animals in quite the elegant fashion. Image from DeAgostini/Getty Images.


Next up is a 1591 illustration from the British Library titled "Newes from Scotland." This image depicts the accused women casting spells over a cauldron. How witchy of them.

Let me begin by stating that the mass persecution of some 30,000-60,000 women and men during the European and Colonial American witch-hunts is a serious and tragic topic that I most certainly won't do justice. If you're looking for a deep dive on witchcraft and the socio-political factors that initiated the hysteria during this era, check out this article (and this)! When researching, I also found this podcast to be extremely informative. To give a brief recount of what I learned, just before the end of the middle ages in Europe (1450's), religious tension, political unrest, and the culturally accepted suspicion and violence towards women led to an increase in witch-hunts across Germany, France, Italy, Switzerland, and the Low Countries. Theological theories of "demonology" fueled these hunts, and those accused were allowed no defensive counsel. This same moral panic infected the lives of Colonial Americans, most notably during the late 17th century witch trials in Salem, Massachusetts.

Surprisingly, of the 144 citizens accused in Salem, none proved to possess any demonic powers. Why then, were they indicted in the first place? When societal unrest reaches its peaks, humanity in turn reaches for control and condemnation to combat the fear and instability of the times. The accused weren't mid-wives or natural healers in their villages, as commonly misinterpreted. Neither were they gathering at witches' Sabbath, or participating in any other mystical rituals. They were normal people, who just so happened to rub someone else in town the wrong way. With that being said, association with herbal remedies and potions were particular past-times that could secure one a spot on the "maybe a witch?" list.

During this period, the general population had a reservoir of plant knowledge that is lost on us today. All medicines and most materials were still derived from plants at the time. Everyone had a grandmother, sister, or cousin, who could prepare a herbal remedy at their bedside if they came down with the sniffles. Plant potions were widely used and normalized ... except when they didn't work. Unfortunately, poor treatment outcomes could lead to herbal practitioners being accused of witchcraft. Imagine telling your doctor "and just so ya know, this rash better clear up soon," with a wink and sly grin as you leave their office. Let's explore some of the plants and remedies used to treat rashes and expel the evil spirits back in the day.

Vervain, Verbena Genus

Being a cosmopolitan genus found in the Americas, Europe, Africa, and Asia, these plants have a deep history in many ancient cultures and traditions. Egyptians believed the plant originated from the tears of the goddess Isis. Others used them to make protective boundaries from evil spirits. Vervain has anti-inflammatory properties which added to its use as a medicinal herb.

Henbane (Hyoscyamus niger)

The Hyosciamus genus contains 17 species, one of which is the black henbane pictured above. Henbane belongs to the family Solanaceae, the nightshades, as do a couple of the other plants I will cover. Historically used for its psychoactive compounds and other healing properties, henbane is now taken as a treatment for intestinal and respiratory issues, tremors, and nausea.

Wolf's bane, Aconitum genus

Native to the mountain ranges of western and central Europe, this lethal plant got its name from its use as a poison frequently added to animal bait. The toxic chemical, aconitine, was also used to coat swords and arrows during wartimes. It would later become associated with witchcraft and murderous potions.

Jimsonweed (Datura stromonium), Datura genus

I present to you another proud Solanaceae member, native to the Americas and now found across the globe. Jimsonweed is known for both its psychoactive and healing properties. During the Spanish Inquisition, use of the plant could lead to immediate prosecution. Currently, Jimsonweed is used to treat asthma, influenza, and nerve diseases.

Mandrake (Mandigora officinarum)

Here's our third representative of the Solanaceae family, the mandrake. This plant belongs to the genus Mandragora, which contains three species native to central Asia and the Mediterranean. Many of you may notice this humanoid looking veg from Harry Potter. If that's not a claim to fame, then I don't know what is.

Rosemary (Rosmarinus officinalis)

Rosemary was sacred to ancient Egyptians, Greeks, and Romans. The Virgin Mary was said to have laid her cloak over a bush while resting, turning the flowers blue and legally changing the plant's government name to "Rose of Mary." It was a tedious process for the plant, but a very relaxing process for Mary.

Mugworts/wormwood, Artemisia genus

The Artemisia genus is quite large, containing 500 species found mostly in North America, Europe, and Asia. Used as a key ingredient in absinthe, a remedy to expel intestinal parasites, and a magical potion conjuring love and protection, this plant served many functions.

Blackthorn (Prunus spinosa)

Native to northern Africa, western Asia, and Europe, this prickly shrub of the rose family has a deep history in Celtic folk-lore. Fairies stood guard at blackthorn thickets and witches used the blackthorn twigs as wands.

catnip/catswort (Nepeta Cataria)

Native to Europe, the Middle East, Central Asia, and parts of China, catnip is used in teas among many cultures for its sedative and healing properties. Also consumed as a juice, tincture, or inhaled smoke, catnip remedied intestinal issues, indigestion, fever, hives, and nervous disorders. The plant's essential oils can be extracted and used as a natural insect repellent. Catnip's tendency to attract cats and pollinators is another plus (unless you're not a cat person).

Belladonna/Deadly nightshade (Atropa belladonna)

The belladonna belongs to the genus Atropa which contains three species native to Europe, Asia, and north Africa. Like the other members of Solanaceae, the plant's psychoactive chemicals were absorbed through ointments rubbed on the skin. In addition to the hallucinogenic and euphoric effects of the belladonna, it (along with all the other nightshades covered earlier) can cause seizures, comas, and even death - so maybe just stick to cocoa butter for your skin hydration needs.

All of the above images were sourced from europeana.eu/en. Check out this site if you're ever looking for images of herbarium sheets or drawings, they have a huge selection.

The grind

October 24th, 2022

Prepare to be be fully submerged into root physiology as we enter the deep-end of our waterlogging project. For months now, you have read update after update on the tissue harvests and photosynthesis measurements we have been taking. The suspense is building and I have received many emails from anxious readers demanding some results. Just kidding, I have received zero emails. If I do, I will surely cherish them, print them out, and stick them to the Root Lab fridge for motivation. In the mean time, I'm motivated purely by the groundbreaking science we're doing here. By "groundbreaking," I mean we literally had to break artificial ground to get to these samples. Keep reading to learn about how we prepped the tissue samples for next week's tests and some background info on assessing stress in plants.

Grinding tissue samples

How do you get over 300 samples containing fine roots, chunky coarse roots, and stems, down to a fine powder? Lots of liquid nitrogen and human-sourced grinding power. Decades down the road, when the Root Lab is automated like the Amazon Fresh grocery stores, there will surely be robots zipping around with mortars and pestles, left with the responsibilities of the monotonous tasks some researchers despise. Personally, I enjoyed the monotony, even though Marvin did most of the grinding (my hands got cold, okay?!).

Let me back up and explain why we needed to grind these samples in the first place. The stress and nonstructural carbohydrate (NSC) tests we will be running on the waterlogging tree samples requires the tissues to be ground to a fine powder in order to homogenize the sample. I will explain the specific chemical markers we're looking at for each stress test later on in this post. For now, let's continue with our tissue grinding protocol.

The samples harvested for this project were immediately frozen in liquid nitrogen, a common procedure taken by researchers collecting any type of live tissue. This helps maintain cell structures and prevents further degradation and/or metabolic processes from occurring in the tissues. Now we have a snapshot of what was going on inside the giraffe, human, or tree, at the time of sampling. When grinding, liquid nitrogen saves the day again by making it easier to pulverize the sample into a powdery substance easily mistaken for different spices and flours. In fact, crushed foodstuffs were the original end products of mortar and pestle use.

The fine root samples, containing the least amount of mass, were ground in full. The roots were dumped from their falcon tubes into a mortar, covered with liquid nitrogen, and then smashed and ground with a pestle. Samples were then transferred back into a falcon tube, weighed, and stored in liquid nitrogen. The same procedure was followed with the coarse roots and stem, except these samples were larger, so we clipped off small portions with pruners prior to transferring them to a mortar.

Lexikon Blogion

Fine roots: the smallest, most distal roots in a plant's root system. We focus on these roots specifically because they are responsible for a plant's uptake capacity. Functions such as water uptake, nutrient uptake, mycorhizzal association, and root exudation, are all mediated through fine roots.

Liquid nitrogen: the most commonly used cryogenic liquid. At a chilly temp of − 320 °F, it is used by scientists for a number of purposes. We use it to preserve and grind samples.

Mortar and pestle: traced back to the stone age, a set of simple tools including a bowl (mortar) and a club (pestle) that are used to grind hard substances into powders and pastes. Tough seeds and grains were probably the first materials ground with the mortar and pestle, but pretty soon the tools' uses and cultural impacts diversified. You can now find a mortar in pestle in the hands of a cosmetologist, artist, chef, pharmacologist, chemist, or anyone else attempting to make guacamole.

Nonstructural carbohydrate (NSC): carbohydrates in plants that are available as a source of energy for growth and other metabolic processes.

Homogenize: to equally distribute or mix. Each of our samples are composed of different tissue layers. We want the chemical analyses we perform to assess the entire sample, not just the bark of the stem or the cortex of the root.

Above you can see our grinding station, equipped with dewars holding liquid nitrogen, a scale, mortars, a pestle, and the mini spatulas/scoopulas that we used to scrape the samples in and out of the falcon tubes.

A fine root sample during and post grinding.

A coarse root sample followed by a stem sample. You can see these samples took an extra stage of chopping before grinding. The fine roots were easier to get down to a powder, but these woodier fellows weren't too difficult to work with - eventually we ground them down.

Stress tests - a final reveal

By now, the stress tests that I've mentioned many times in previous posts may seem like a perpetual mystery that will never be explained. It was all a ploy to keep you coming back for more! Hopefully the pay off is as thrilling as Bruce Willis's character discovering he's been a ghost THE WHOLE TIME. For those of you who haven't seen The Sixth Sense, Willis, assuming the role of a psychologist, tries to help a young patient cope with his visions of the dead. I won't spoil the ending for you here (if you want spoilers, shift your eyes up about three lines of text). I know, I know, "what does this have to do with roots?!" Everything, actually. This research was funded by Mr. Willis himself.

To transition away from my poor attempts at sarcasm that are probably difficult to read over the internet, let's move on to the tests we're running on our fine root samples to assess their stress levels and the cellular damage they accrued over the course of the experiment:

Peroxidase activity
Peroxidase is a ubiquitous
enzyme found throughout the bacterial, animal, fungal, and plant kingdoms. In plants, these proteins are involved in many processes of growth and development. Our leafy friends have multiple types of peroxidases (just like we do) that are up- and down-regulated in response to various stimuli. Previous research shows that introduction of some stressors (e.g. herbivory, heavy metal pollution, excessive heat), impacts the activities of these peroxidases differently. These proteins are known as stress enzymes because they serve the essential function of degrading reactive oxygen species (ROS). ROS are natural byproducts of the chemical reactions that occur within cells to create energy, otherwise known as cellular respiration. The production of ROS increases under stressful conditions, such as waterlogging, and their build up leads to cellular damage and inhibition of many cellular functions. Enough ROS induced damage will eventually cause tissue death, as seen in the root die-back of our intolerant tree species. Check out this in-depth review on plant peroxidases if you want to learn more about their functions and applications.

We will test the peroxidase activity in our fine root samples by using a reagent containing the compound guaiacol. Guaiacol is helpful substrate to use when quantifying peroxidase activity because it turns the sample to a brownish color that can then be measured spectrophotometrically. When peroxidase is present in a sample, it catalyzes the following reaction:

2 H2O2 → 2 H2O + O2

For anyone who is not fond of chemical equations, no worries. Basically, peroxidase is speeding up the decomposition of hydrogen peroxide, and producing water and oxygen. That lonely diatomic oxygen molecule you see towards the end of the reaction combines with guaiacol to form a brown color. Higher peroxidase levels lead to quicker production of oxygen. The more oxygen in the sample, the more bonds guaiacol can form, producing darker brown hues.

What can we expect to see in our trees? We're not sure, but we have some ideas. The tolerant trees that showed little root die-back, i.e. the silver maples, could be synthesizing more peroxidase proteins in order to scavenge the ROS produced by the stress of waterlogging. Intolerant trees may show high peroxidase activity as well, and that would tell us that having more scavengers wasn't enough to mitigate tissue damage and save the tree.

Lexikon Blogion

Peroxidase: a large group of enzymes involved in many cellular processes. The peroxidases found in plants are defined as class III peroxidases. Within that class are different forms of peroxidase with unique structures and functions. Genetic research has found that the number of genes coding for these enzymes vary between species of plants. In rice, 42 members of the class III peroxidases have been sequenced. Numbers are closer to a dozen in tobacco plants.

Enzyme: proteins produced in all living organisms that catalyze, or speed up, biochemical reactions. Technically, there are also some non-protein enzymes, such as ribozymes, which are ribonucleic acid enzymes. This is a rabbit ribo-hole topic for another time!

Reactive oxygen species (ROS): an unstable molecule that readily reacts with lipids, proteins, and DNA. ROS, often called 'radicals,' are reactive because they contain one or more unpaired electrons. These sneaky thieves zoom around, stealing electrons through a process called oxidation.

Spectrophotometry: a branch of electromagnetic spectroscopy that measures the number of photons emitted vs. absorbed by a sample.

Lipid: a class of organic compounds composed of fatty acids and their byproducts. These include fats, waxes, oils, and steroids, to name a few.

Oxidative damage: imbalance that occurs within a biological system when the amount of ROS exceeds the ability of the organism to detoxify and/or repair the damage caused by the radicals.

Oxidize: a chemical reaction which causes electrons to be transferred to another molecule.

Lipid oxidative damage

Remember all of that cellular damage I was talking about earlier? The lipids that form a plant's cellular membrane are the unlucky receivers of such harm. To throw in a sports reference for all the sporty readers out there, lipid membranes are like the offensive line on a football team. ROS are the defensive players on the other team, continuously pushing back against the offensive line. When the offensive line is strong and intact, they form a barrier and protect their key players. Tom Brady can serve as the nucleus for this metaphor, and some running-back (apologies, I don't know the names of any current running-backs) can play the role of another organelle, the mitochondria. When the lipid membrane is weakened by too much ROS damage, Tom Brady gets sacked or the anonymous running-back gets tackled behind the line. According to football fans, that's bad (if it's your team).

I'll throw in some microbiological explanations for all the microbiology-y readers out there. Lipid peroxidation, the chain reaction caused by lipid oxidative damage, is a common biomarker used to assess stress and cellular damage in plants, animals, bacteria, and so on. When ROS oxidize the unsaturated fatty acids that form the lipid membrane, this deleteriously impacts the membrane's properties, leading to protein degradation, limitation of ionic transport in and out of the cell, and ultimately cell death. According to cell fans, that's bad.

The cellular membrane destabilizes and becomes permeable as a result of oxidative damage. Image source

To test the levels of lipid oxidative damage in our fine root samples, we will be using thiobarbituric acid-reactive substances (TBARS). When lipid peroxidation occurs, one of the byproducts is a chemical called malondialdehyde (MDA). MDA reacts with the acid in the TBARS test to form a pinkish color. We can then quantify the damage by measuring the sample spectrophotometrically, just like the previous peroxidase activity test. If the root's offensive line really took a beating, there will be a darker color change.

The oxidative damage is expected to be much higher in the waterlogged sugar maples compared to the controlled counterparts. They experienced a fair amount of root die-back, which is assumed to be influenced by high lipid oxidative damage. As for the rest of the species, predictions for both low and high levels seem sensible. The silver maples held onto their roots, but it's possible that a lot of internal damage was endured and the samples could still contain high amounts of MDA. Both magnolias experienced root die-back followed by a new flush of growth during the recovery period. Will some older age classes (pre-waterlogging) express higher damage, while the younger classes (post-waterlogging) show no damage at all? We'll find out soon enough.

Special shout out to Dr. Chad Rigsby and Dr. Carla Rosenfeld who have both been a huge help in deciding on protocols and running these tests!