Messages from the mycorrhizas

Banner art by Agorastos Papatsanis

As the sunlit hours decrease day by day and we creep slowly towards the darkest season in the Northern Hemisphere, it is easy to assume that nature, and our own wonder, have gone dormant. All it takes to disabuse one from such a belief is a closer look, underneath a blanket of what appears still and inanimate. To further encourage this shift in focus, I'll call upon reoccurring blog guest, Emily Dickinson, and her poem "Before the ice is in the pools."

Before the ice is in the pools—

Before the skaters go,

Or any check at nightfall

Is tarnished by the snow—

Before the fields have finished,

Before the Christmas tree,

Wonder upon wonder

Will arrive to me!

This week, in my own attempt to maintain wonder, I explored the magical world of fungi by investigating mycorrhizal signaling and the teeny-tiny proteins responsible for some of Earth's most important symbiotic relationships. I also played around with R and plotted the leaf data from our waterlogging project. Keep reading to find out just how many leaves fell and how mycorrhizae use peptidal communication strategies to maintain healthy bonds between fungi and host plants.

Waterlogging leaf data

One of the measurements used to assess tree health and performance during the waterlogging study was leaf counts. Similar to the assimilation rates we measured to assess the photosynthetic function of the trees, leaf counts provide important data that will help tie our belowground findings to aboveground impacts. Leaf loss is a common response in trees that are poorly coping with waterlogging stress, making it an important marker for species' tolerance levels. Remember, the deep and burning question we are addressing in this study is how root responses to waterlogging influence whole-tree response and tolerance.

In the above graphs you can see how the leaf counts of each species fluctuated over time and reduced/increased from the original leaf count number (shown as 100%). Generally, all species controls maintained higher counts than the treated trees. The silver and sugar maple counts support what we know about the difference in tolerance levels between maple species. The silvers lost few leaves because they were able to tolerate the waterlogging stress and the sugars leaves dropped dramatically as they struggled to maintain normal physiological functions during waterlogging. Both magnolia species control counts aren't necessarily what we would expect. This probably has more to do with the fact that some of our magnolias, especially the star species, had immature and burned leaves at the beginning of the project which may have resulted in premature leaf loss regardless of the treatment. For now, we'll continue to wonder about what these results tell us.

Mycorrhizal Messaging

The foundation of every relationship is communication. Even amongst nature's trendiest (and oldest) celebrity couples, such as roots and fungi, signals must be sent and received before any lasting partnership can occur. When the first rootless plants began their migration onto Earth's terrestrial landscape during the Ordovician period 475 million years ago (Mya), fossil records show that fungi were evolving alongside them, forming the earliest versions of the mycorrhizal symbioses we study and admire today. Scientists initially believed that species within the subphylum Glomeromycotina were the only fungi forming mutualistic relationships with ancient plants, but recent research has hinted at a more complex story. More species within the larger Mucoromycota clade are now thought to share a symbiotic history with early non-vascular and vascular land plants, as reviewed by Hoysted et al. here. Regardless of their phylogenetic origin, what all these fungi have in common is their ability to both send and receive signals from their plant partners. These signals have evolved through time, just as human technology has transitioned from the telegraph to the sleek little box of unlimited potential we all have in our pockets now. Fortunately for fungi and plants, their communication devices won't submit them to awkward group texts or ads for mud coffee.

Before proceeding any further, it would be wise of me to introduce and describe the fungal crew I'll be referring to throughout this segment. In a previous post, I covered arbuscular mycorrhiza (AM) and ectomycorrhiza (EM) fungi. However, the connections they form with their host roots are much more complex and nuanced than I originally let on. Fungi that form AM associations, along with ericoid mycorrhizas and orchidaceous mycorrhizas, are within a larger group known as endomycorrhiza. These differ from EM types by their methods of root tissue colonization and soil exploration. Endomycorrhizal fungal tissue is mostly housed within the root host, whereas ectomycorrhizal types form a mantle on the outside of roots and have extramatrical hyphae extending beyond their root host and into the soil. Fungi that form EM associations, including all the mushroom species introduced in previous blog entries, also incorporate a smaller subgroup called ectendomycorrhiza. As you can probably guess by their extremely fun to say name, ectendomycorrhizas have characteristics of both ecto- and endomycorrhizas. They are a choosy bunch, only associating with the genera Pinus (pine), Picea (spruce), and Larix (larch). The image below depicts all of the above mycorrhizal types and their means of host root colonization. To find out more about mycorrhizal biology, check out this site.

Now that you're all familiar with the leading cast of this mycological movie, let's dig into the relationship dynamics between fungi and plants. As I mentioned earlier, the symbiosis between fungi within the clade Glomeromycotina and their plant hosts has been widely recognized as one of the most ubiquitous mutualisms found in nature. Over 80% of land plants are thought to form AM associations, and many of these plant hosts include important food crops. In fact, farmers and researchers have used AM inoculated soils to increase crop yields of grains and vegetables. The outcomes of these relationships are well known: plants are supplied with phosphorus, nitrogen, water, and in some cases a higher resistance to other pathogens and environmental extremes, as courtesies of their fungal partners. In return, fungi receive a payment of around 10-20% of the plant's total photosynthate production. The amount of nutrient and sugar exchange vary widely and depend on many variables such as the species involved, environmental conditions, seasonal fluxes, and so on. These results are relatively easy to observe and measure when compared to the tiny signaling molecules that initiate this affair. So, who made the first move?

Disclaimer: I want to make it clear that my anthropomorphizing of the characters in this film should in no way be taken seriously. My aim is to make these posts enjoyable, but also to tell a story that is easy to understand and recall at later times. If a stranger next to you cries out: "UGH, if only I understood mycorrhizal associations, then all of my problems would be solved and I could save the world," it would fill me with joy to know that you could solve that stranger's highly specific problem. This is all to say, mycorrhizal associations aren't driven by any conscious decision of the species involved. The nutrients exchanged between organisms aren't traded on a one-to-one ratio. Fungi aren't FedEx-ing phosphorus to the plant and waiting for a payment in the form of sugar or Bitcoin. This is a highly dynamic system where many things are regulated simultaneously. Both fungi and plants have evolved to work together, but they have also evolved for survival and replication of their individual genes and species.

Back to our regularly schedueled programming on the molecular dialogue that's orchestrating the symbiosis between fungi and plants. Before fungi colonize their host plant's roots, they need to ensure that they have adequate space and a specific type of tissue in which they can house their hyphal webs. To investigate the steps of mycorrhizal colonization, Gutjahr et al. performed an experiment on rice plants and AM fungi and found that fungal signaling initiated the growth of lateral roots, which have a larger diameter and more cortical tissue. This is important because the cortex is the area of the root colonized by AM fungi. By showing that the architecture of the rice roots was modified before penetration of the fungal hyphae, scientists concluded that their must be signaling during the pre-symbiotic stage to prime the host root and initiate root growth.

Below you can see images of AM root colonization from Yuan and colleagues. Roots and fungi are stained using a blue dye, the chitin in the fungal cell walls retains the blue color, allowing us to examine the filamentous hyphae (string-like cords) and vesicles (orbicular structures) formed from fungal tissue.

While specifics regarding the fungal signaling molecules that cause such phenomena like the lateral root branching described above remain mostly unknown, there has been some headway into isolating and identifying these pre-symbiotic compounds. Chitooligosaccharides have been of particular interest to researchers and have a variety of applications. They are found to promote mycorrhizal symbioses by suppressing plant host immunity and initiating changes in root metabolic pathways and tissue development. These chitin-based compounds have even been isolated from commercial shrimp fisheries and fungal sources to use as agricultural biofertilizers. Current research is being done to find sustainable ways to isolate chitooligosaccharides from large-scale mycelial production. This is one example of how fungi are proactively sending signals into the environment while house-hunting for their future living quarters. What are plants doing to ease the move?

In the last blog update, I mentioned branching factors and strigolactones, which are a class of compounds found in root exudates that facilitate mycorrhizal colonization. For quite some time, the impacts of these mysterious root compounds on hyphal branching were observed and known. Due to the extremely low concentrations produced by plants and their chemical instability, they have been tricky to isolate and identify. Exciting work in the last two decades has identified the chemical structures of strigolactones and elucidated their roles in both mycorrhizal development and other ecosystem relations. One important driver of strigolactone excretion seems to be phosphorus depletion. From what we know about mycorrhizal fungi and their nutrient scavenging abilities, this makes perfect sense. If a plant is unable to meet their phosphorus needs, why not exude an itty-bitty compound into the soil and propose to a fungal stranger: "I will let you move in if you pay me rent in phosphorus." However, as yet another reminder of how ecology and interactions are never that simple and romantic, this strategy doesn't always benefit plants. Seeds of parasitic weeds also germinate in response to strigolactones and can have devastating impacts on crops such as legumes, sunflowers, tomatoes, tobacco, and many cultivated cereals. Below is a diagram depicting how this process works.

Discovery of chitooligiosaccharides, strigolactones, and other compounds involved in mycorrhizal signaling have helped inform models for fungi-host interactions and the physiological changes that occur in both symbionts. Colonization occurs in stages: pre-symbiotic compounds are released by roots and fungi, first contact between root and fungi induces hyphae to differentiate into hyphopodium, a penetration hyphae emerges from the hyphodium and invades the rhizodermal cell layer, and the last stages concerning how the fungus spreads within the root depends on the type of mycorrhizal fungi involved. If the new tenant is from the endomycorrhizzal group covered earlier, the fungus will spread longitudinally within the cortex and form arbuscules inside the plant's cortical cells. If ectomycorrhizal, the fungus won't penetrate deeply into the root cell, and instead puts its energy into forming a mantle of hyphal cells covering the outside of the root, and Hartig net hyphae inside the root. From what I could find when doing research for this post, the depth of penetration of the Hartig net depends on the species involved. For angiosperm associated mycorrhizas, it seems the Hartig net doesn't stray far from the root's surface and forms a network between the uppermost epidermal cells. As for gymnosperm associates, the Hartig net can extend deeper into the root, branching further down between cortical cells like the following image shows. While we're on the topic of EM interactions, let's look at research that has focused on their pre-symbiotic signals and the physiological impacts on their root symbionts.

Reminiscent of the experiment described earlier investigating the impacts of AM fungal signaling on rice plants, Felton et al. tested an EM species with poplar trees and found similar results. Prior to physical contact with poplar roots, fungal signals initiated lateral root formation in the trees. The study was taken a step further by analyzing gene expression and hormone levels within the root tissue. Fungal compounds increased auxin levels in the apex of the root while also increasing apical and lateral polar auxin transport. Not only did these exogenous fungal compounds increase plant growth hormone activity, they also impacted their transport through the plant, leading to the initiation and growth of lateral roots. What's even more interesting are the broad effects of these signaling molecules. Felton and colleagues also monitored the changes induced in non-mycorrhizal plants and found that they experienced the same lateral root formation. Recent developments have dug deep to identify where the genes for these compounds exist within the fungal genome in an effort to describe their function and evolution more clearly.

These images show an increase in lateral root formation in poplar seedlings as mycorrhizal colonization increases. Scientists used a dual staining method which dyes the fungal cells green and the plant cells magenta.

In 2008, more than a decade after its fungal kingdom cousin Baker's yeast (Saccharomyces cerevisiae) was sequenced, Laccaria bicolor became the first multicellular fungus to have its genome sequenced. The bicolored deceiver is a cosmopolitan species that forms associations with many EM plant communities, making it an ideal model organism for fungal studies. In fact, the paper referenced above chose L. bicolor as its EM representative, and because of my strong intuition/psychic powers, I can confidently proclaim that the following publication you'll see summarized will also involve the fibbing fungi. When analyzing the genetic makeup of this common mycorrhizal mushroom, Martin and colleagues finally put a face to the name in regards to those mysterious signaling molecules I've been baiting you with for the duration of this post. The genome also told an interesting story involving high amounts of repeated sequences and the loss of some carbohydrate-active enzymes, but we'll cover that in the following section. Back to the signaling molecules, mycorrhizal fungi are known to release tiny compounds into the soil and their host plant's tissue called mycorrhiza induced small secreted proteins (MiSSPs). Martin found evidence of the regions that code for MiSSPs, but the exact function of these proteins were unknown at the time. It was also shown that a proportion of these regions were only expressed in symbiotic tissues, a finding that Pereira et al. would extrapolate on in a 2018 study analyzing the secretome of another common EM species, Cenococcum geophilum.

Cenococcum geophilum is one of the most well-studied fungal species because of its abundance and wide distribution in forest ecosystems, and therefore has a lot to tell us about mycorrhizal symbioses. Pereira and crew found over 200 MiSSPs involved in the interactions between C. geophilum and two different plant hosts, pine and poplar trees. Most of these gene regions and the MiSSPs they code for showed the same levels of regulation regardless of the host. This provides insight into C. geophilum's symbiotic strategy - the signaling molecules secreted aren't host specific. However, the opposite has been shown in other mycorrhizal fungi. Some may only associate with one plant species. Others will regulate the production and secretion of different MiSSPs depending on what plant host they are associating with. Returning to Martin's earlier findings of repeated sequences and the loss of carbohydrate-active enzymes, C. geophilum's genetic code narrated a similar story.

What are repeated sequences and what do they tell us about the evolution of mycorrhizal fungi? Envision flipping through the pages in a book and finding a paragraph full of repeating A's, C's, G's, and T's. After a couple indecipherable lines of text, the writing returns to normal and your pleased to find that your book wasn't written in some secret code. You flip the page again only to come across an entire chapter of these random four letter repeats. Although you can't make any sense out of what seems to be a mishap of the publishing company, what you're looking at isn't an error nor evidence of the author's cat walking across their keyboard. It serves a purpose! Okay, my metaphor falls short here, because I can't come up for any logical reason why an actual book would contain repeated strings of letters, BUT, in the case of DNA and genomes, this a common occurrence. When we sequence a species genome, we are essentially reading the genetic biography of that species, and the language in which the book is written is in base pairs of DNA or RNA. Scientists have questioned the function of repeated sequences, which typically don't code for proteins (or, using my earlier example: words), and now believe they play important roles in expression and regulation of other genes. In the case of fungi, repeat rich regions have been found to evolve at faster speeds compared to other regions of their DNA. This allows for higher mutation rates and subsequent gain and/or loss of genes that assist fungi in interacting with their plant hosts.

Above you can see the smallest units of DNA, nucleotides (bases), and the hydrogen bonds they form with their complimentary base. For further explanation look to the glossary.

When looking at the evolutionary history of EM fungi, their story traverses from lone wolf to symbiotic significant other. Many genetic lines of EM fungi have convergently evolved from species of saprotrophic fungi. Through this slow and steady change, they have lost genes for decaying organic matter and gained genes for coding diffusible signaling molecules. In other words, they lack the carbohydrate-active enzymes involved in degradation of plant cell walls and maintained the ability to degrade non-plant cell walls, leading to a dual saprotrophic and biotrophic lifestyle. This dual approach allows them to live freely in the soil, feasting on soil microbes, until they are able to associate with a viable plant host. In contrast, as was covered earlier, AM fungi are obligate symbionts. A plant partnership is needed for them to meet their nutrient needs and complete their lifecycles. One can imagine the divergent evolutionary paths this "lifestyle choice" would lead both groups down. Analyzing the similarities and differences in the genomes of these fungal groups sheds a light on their evolutionary past and the strategies they use to interact with their host plants.

We have covered the epic partnership between fungi and plants, but they weren't always nature's it couple. This symbiosis can be found in virtually every nook and cranny of the Earth's terrestrial biosphere and there remains much to learn about the mycorrhizas in our own back yard, let alone the undiscovered ones across the globe. Research focusing on signaling molecules and the subsequent physiological changes that promote and maintain symbiosis between fungi and plants will not only provide us with mechanisms for how this interaction takes place, but also offers applications to improve the health of different ecosystems and increase crop yields. Both science and the public's current interest in belowground ecology pushes such research forward. When we maintain wonder and reverence for the life beneath our feet, slowly but surely more is revealed to us.

Lexikon Blogion

Ordovician: A 40 million period within the Paleozoic Era beginning 485 Mya as the Cambrian Period came to a close and ending 443 Mya, prior to the start of the Silurian Period. Most notably for us plant people, the first land plants made their journeys up the sandy shores during this time.

Glomeromycotina: one of eight divisions in the kingdom fungi containing over two hundred known AM species. Fungi in this group are obligate symbionts, meaning they need to associate with hosts to meet their energy demands and to synthesize materials such as the lipids that construct their cell walls. Forming associations with ~80% of land plants, Glomeromycotina is one of the most ecologically important fungi divisions. However, they are extremely difficult to study. Only some species can be cultured under lab conditions, leaving scientists to rely on molecular markers as identification tools for this group.

Mucoromycota: The larger fungal clade that Glomeromycotina, along with two other subphyla: Mortierellomycotina and Mucoromycotina, is grouped under. Most of its members are mycorrhizal, endophytic, or plant matter decomposers.

Phylogeny: We've covered taxonomy and systematics previously, but as a refresher - phylogeny is the study of the evolutionary history of a species and their relationship within and between groups.

Ericoid mycorrhiza: The association formed between fungi within the phylum Ascomycota (sac fungi) and plants in the order Ericales (heath, heather, blueberry, etc). These endomycorrhizal fungi assist their plant hosts in colonizing the harsh environments and acidic soils of moorlands, which can be found in upland regions of temperate grasslands. One last fun fact: the fungi in this group are facultative symbionts, this means that their mycelia can live freely in the soil, feeding saprotrophically, when not paired with a plant host.

Orchidaceous mycorrhiza: An association formed between fungi within the phylum Basidiomycota (club fungi) and plants in the family Orchidaceae - the largest family of flowering plants with over 22,000 confirmed species. During their embryonic stage and first years of life, orchids are non-photosynthetic and rely completely on their fungal partners for nutrient and energy sources. As the plants mature, their reliance on mycorrhizal nutrient exchange can wax and wane, and the fungal partner may even be dumped for a completely different species. Interestingly, many of the EM fungi orchids associate with are highly parasitic towards other plants. You could say that orchids have a bad boy complex.

Cortex: Formed by layers of undifferentiated cells in the stems and roots of plants, the cortex is located between the epidermis (outer cell layer) and the vascular or conducting cells. Most of the cortical space is filled with thin-walled parenchyma cells, while the outer layer is composed of thicker collenchyma cells. Water and nutrients reach the plant's inner transport tissues by diffusion through the cortex. This area also allows for energy storage in the form of starch.

Chitooligosaccharides: Chains of simple sugars that are produced through the enzymatic breakdown of chitin. Chitin can be found in the cells walls of fungi and the exoskeletons of arthropods. It is also produced by some bacteria.

Hyphal branching: The process by which filamentous fungi are able to grow, spread, and increase surface area for higher nutrient acquisition. There are two modes of branching: apical and lateral. When branching occurs at the fungal tips it is deemed apical, when branching initiates from sub-apical hyphal compartments, it is lateral. Most branching occurs laterally, with new branches growing away from neighboring branches in order to cover the most soil area as possible.

Hyphopodium: a hyphal branch specialized for the function of nutrient absorption, typically composed of one or two lobed cells.

Rhizodermis: a fancy word for root skin. This is the outermost cell layer of the root. Most plants have specialized rhizodermal cells that form structures called root hairs and trichomes.

Arbuscule: the site of nutrient exchange between AM fungi and their plant hosts. These intricately branched hyphae form tree-shaped structures with large surface areas inside plant cortical cells. Gutjahr and Parniske have reported that lifespans of individual arbuscules span a short period of 1-3 days.

Mantle: a sheath or sock-like covering formed by the hyphal tissue of EM fungi that covers the host plant's fine roots.

Hartig net: the network of penetrating hyphae that grows through the host plant's epidermis and between the root cortical cells. The Hartig net is the site of nutrient exchange for EM species. Fun fact: the net gets its name from 19th century German botanist Theodor Hartig who researched the anatomy of the EM/plant host interface in the 1850's.

Auxin: a class of plant hormones responsible for the regulation of growth and development. Auxin's mode of action is to loosen cell walls, allowing them to elongate and expand.

Secretome: the totality of proteins secreted by a cell, tissue, or organism. FYI, this isn't only an inquiry into fungi, plants, slugs, or any other species you may think of when you hear the word "secrete." According to the Human Protein Atlas, 36% of human protein-coding genes are predicted to interact with cell wall transporters, meaning they will meet the ultimate fate of being secreted out of your cells and/or body.

Base Pairs: the fundamental unit of double-stranded nucleic acids consisting of one purine bonded to its complimentary pyrimidine. In DNA, purines consist of adenine (A) and guanine (G), and pyrimidines include thymine (T) and cytosine (C). You can think of each base pair forming a rung of a ladder, if the ladder was a DNA molecule. Purines and pyrimidines are connected by a hydrogen bond, always forming the following pairs: AT and GC. The same goes for RNA except the pyrimidine thymine is replaced by uracil (U).

Convergent evolution: the development of similar form and/or function between different species due to the same selective pressures on both species. This will often lead to species occupying the same ecological niches such as habitat, resource use, and their relations with abiotic and biotic factors in their community. In other words, convergent evolution is not based on genetic relatedness. Many different species of EM fungi have evolved the ability to synthesize signaling proteins and have lost the ability to degrade plant cell walls. This isn't because of their distant family lineage, but rather through the evolutionary forces placed upon them in similar soil environments.

Saprotrophic: commonly used to describe fungal and bacterial feeding habits, saprotrophy is the process of extracellular digestion of decaying organic matter. In simpler terms, fungi and bacteria excrete a bunch of digestive enzymes into the soil which breaks down the dead organic matter into simpler compounds that they can then absorb.

Biotrophic: a relationship in which a parasitic organism feeds and reproduces on its host without killing it. Some examples include plant pathogenic bacteria, fungi, and nematodes.